Note: Descriptions are shown in the official language in which they were submitted.
CA 02173156 2004-11-01
WO 99110034 217 3 i S 6 pCTNS94111037
AMTSOb FOEt CRAAACTE1tIZIW POLYMER MOLEC'GL88 OR TBE LISB
FIgLD OF TSg INVSNTZON
This invention, in the fields of electrophoresis,
microscopy, spectroscopy and molecular biology, relates to
methods for characterizing polymer molecules or the like, for
example, observing and determining the size of individual
particles and determining the weight distribution of a sample
containing particles of varying size. More particularly, this
invention involves the use of microscopy and/or spectroscopy in
combination with spectroscopic methods to characterize
particles, e.g., nucleic acids, such as by measuring their
positional and conformational changes when they are subjected to
an external force, auch as a restriction enzyme digest and by
measuring their length and diameter or radius.
BAC~G~tOOND OF T88 IDTVmITIOli
Traditionally, the molecular weight distribution of a
sample of particles has been determined by measuring the rate at
which particles which are subjected to a perturbing force move
through an appropriate medium, e.g., a medium which causes the
particles to separate according to size. A mathematical.
relationship is calculated which relates the size of particles
and their migration rate through a medium when a specified force
is applied.
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Sedimentation is a well-known technique for measuring
particle size~, but, when applied to polymers, this method is
limited to molecules with a maximum size of about 50=100
kilobases (kb). Attempting to measure larger molecules by this
technique would probably result in underestimation of molecular
size, mainly because the sedimentation coefficient is sensitive
to centrifuge speed. See Kavenoff et al., Cold Sprlag Harbor
Symp. Quantit. BloI., 381 (1974)).
Another popular method of separating polymer particles
by size is by gel electrophoresis (see, e.g., Freifelder,
Physical Biochemistry, W.H. Freeman (1976), which is
particularly useful for separating restriction digests. In
brief, application of an electric field to an agarose or
polyacrylamide gel in which polymer particles are dissolved
causes the smaller particles to migrate through the gel at a
faster rate than the larger particles. The molecular weight of
the polymer in each band is calibrated by a comparison of the
migration rate of an unknown substance with the mobility of
polymer fragments of known length. The amount of polymer in
each band can be estimated based upon the width and/or color
intensity (optical density) of the stained band. However, this
type of estimate is usually not very accurate.
Pulsed field electrophoresis, developed by the present
inventor and described in U.S. Patent No. 4,473,452,
is an
electrophoretic technique in which the separation of large DNA
molecules in a gel is improved relative to separation using
conventional electrophoresis. According to this technique,
deliberately alternated electric fields are used to separate
particles, rather than the continuous fields used in previously
known electrophoretic methods. More particularly, particles are
separated using electric fields of equal strength which are
transverse to each other, which alternate between high and low
intensities out of phase with each other at a trequency related
to the mass of the particles. The forces move the particles in
an overall direction transverse to the respective directions of
the fields. It should be noted here that the term "transverse"
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as used herein is not limited to an angle of, or close to, 900,
but includes other substantial angles of intersection.
One of the most significant problems with determining
the weight of molecules by indirect measurement techniques, such
as those described above, is that the parameters which are
directly measured, e.g., migration rate, are relatively insensi-
tive to small differences in molecular size. Thus, a precise
determination of particle size distribution is difficult to
obtain. The lack of precision may particularly be a problem
when biological polymer samples, which tend to be unstable and
contain single molecules inches in length, are involved.
While some of the known methods of determining
particle size distribution in a polydisperse sample provide
better resolution than others, few, if any, of the previously
known techniques provide resolution as high as is needed to
distinguish between particles of nearly identical size. Gel
permeation chromatography and sedimentation provide resolution
of only about M'/2 (M=molecular weight). Standard agarose gel
electrophoresis and polyacrylamide gel electrophoresis provide
resolution varying as -log M. Pulsed electrophoretic techniques
are effective for separating extraordinarily large molecules,
but do not provide much better resolution than standard
electrophoresis. Thus, the ability to distinguish between
particles of similar size, for example, particles differing in
length by a fraction of percent, is inaccurate and problematic
using the above-described measurement techniques.
Particles of higher mass (i.e., up to approximately
600 kb) can be resolved using conventional gel electrophoresis
by reducing the gel (e.g., polyarylamide) concentration to as
low as 0.035% and reducing field strength. However, there are
also problems with this method. Most notably, the dramatic
reduction in gel concentration results in a gel which is
mechanically unstable, and less sample can be loaded. An
electrophoretic run to resolve very large DNA molecules using a
reduced gel concentration and field strength may take a week or
more to complete. Furthermore, a reduced gel concentration is
not useful to separate molecules in a sample having a wide range
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of particle sizes, because separation of small molecules is not
achieved. Thus, if a sample containing molecules having a wide
range of sizes is to be separated, several electrophoretic runs
may be needed, e.g., first, a separation of the larger molecules
and then further separation of the smaller molecules.
Other particle measurement techniques known in the art
are useful for sizing certain molecules which are present in a
bulk sample, (e.g., the largest molecules in the sample, or the
average molecular size) but are impractical for measuring many
polymers of varying length in a given sample. The viscoelastic
recoil technique, (see Kavenoff et al, "Chromosome-sized DNA
molecules from Drosophila," Chromosoma 411 (1973)) which is well
known in the art, involves stretching out coiled molecules in a
solvent flow field (e.g., a field which is created when fluid is
perturbed between two moving plates) and determining the time
required for the largest molecule to return to a relaxed state.
Relaxation time is measured by watching the rotation of a
concentric rotor which moves during the time of relaxation.
While this technique is quite precise in that sample determina-
tions vary as M'-" when applied to large DNA molecules, it is not
useful for sizing molecules other than the largest molecule in
the sample.
Using light scattering techniques, which are known in
the art, (e.g., quasi-elastic light scattering), the size and
shape of particles are determined by a Zimm plot, a data
analysis method which is known in the art. With these
techniques, size dependence varies as M'. Light scattering
requires that the solution in which the molecules to be measured
are placed is pure, that is, without dust or any other
contamination, and it is therefore unsuitable for sizing a DNA
sample. Furthermore, it is not useful for sizing molecules as
large as many DNA molecules, and is useful only for determining
the average weight of particles in a sample, not the weight
distribution of a sample with particles of various sizes.
Yet another particle measuring technique which is
known in the art for measuring individual molecules provides
measurements of particle size having limited accuracy. The
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average size and shape of individual, relaxed DNA molecules has
been determined by observing the molecules under a fluorescence
microscope, and measuring the major and minor axes of molecules
having a spherical or ellipsoid shape (see Yanagida et al, Cold
Spring Harbor Symp. Quantit. Biol. 47177, (1983)). This
technique is performed in a free solution, without perturbation
of the molecules.
The movement of small DNA molecules during electropho-
resis has been observed (see Smith et al. Science. 243203
(1989)). The methods disclosed in this publication are not
suitable for observation of very large DNA molecules, and
techniques for measuring molecules are not discussed.
Practical weight determinations of particles such as
polymer molecules depend not only upon maximizing the size
dependencies of the directly measured parameters, but also upon
factors such as the amount of sample needed, the time required
to complete an analysis, and the accuracy of measurements. Gel
permeation chromatography can be time-consuming and requires a
large amount of sample. Methods such as conventional gel
electrophoresis can be relatively time-consuming, require
moderate amounts of sample, and cannot size very large DNA
molecules.
Molecular sizing is a fundamental operation that
touches virtually every aspect of genomic analysis from DNA
sequencing to size measurements of lower eucaryotic chromosomal
DNAs. Molecular size, given in kilobases, can be translated
into centimorgans for many organisms, and vice-versa; and gel
electrophoresis is generally used to determine these sizes. The
basics in nucleic acid sizing technology, as practiced by the
typical molecular biologist, have not changed very much in the
past decade. This is understandable considering the simplicity
of gel electrophoresis and its capacity for parallel processing
of multiple samples. The data obtained from gels are readily
interpretable. Given the size of most genes, gel
electrophoresis techniques adapt well to their analysis. From
characterization of restriction digests to discernment of one
base differences in sequencing ladders, gel electrophoresis is
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1'~315~
the method of choice ~ or size analysis of DNAs. Even the
outcome of PCR (Mullis, Methods in Enzymol, 155335-350 (1987))
reactions is frequently monitored by sizing analysis. Pulsed
gel electrophoresis extends this coverage even further to
include chromosomal DNAs from lower eucaryotes (Schwartz, Cold
Spring Harbor Symp. Quant. Biol., 4789 (1983); Schwartz Cell,
3767 (1984); Carle, Nucleic Acids. Res. 125647 (1984); Chu,
Science 2341582 (1986); Clark, et al., Science 2411203 (1988).
Because pulsed electrophoresis can resolve very large DNA
molecules, its application has simplified the mapping of large
genomes and provided a necessary tool for creating large YACs
(yeast artificial chromosomes) (Barlow, et al., Trends in
Genetics 3167-177 (1987); Campbell, et al. Proc. Natl. Acad.
Sci. 885744 (1991)). However, pulsed electrophoresis was
developed more than 10 years ago (Schwartz, Cold Spring Harbor
Symp. Quant. Biol. 4789 (1983) and Schwartz Cell, 3767 (1984)).
The surprising lack of significant sizing advances is contrasted
to progress made in understanding the molecular mechanisms of
conventional and pulsed electrophoresis (Zimm, Quart. Rev.
Biophys. 25171 (1992); Deutsch, Science 240992 (1988).
Although molecular size determination has not advanced
significantly in this decade, another aspect of genomic
analysis, DNA detection technology, has progressed to a
remarkable extent. These developments have impacted on gel-
based methodologies as well as on the field of cytogenetics. A
driving force has been the Human Genome Initiative and its goals
to characterize the human genome and the genomes of model
organisms by extensive mapping and sequencing. The new goals
aimed at analyzing entire large mammalian genomes include
increasing accuracy and high throughput of DNA mapping and
sequencing. The first round of needed advances has come in part
from a combination of sophisticated image processing methods
(Glazer, Nature 359859 (1992); Quesada, BioTechniques 10616
(1991); Mathies, Nature 359167 (1992)); new DNA detection
techniques and new DNA labeling/imaging systems (Glazer, Proc.
Natl. Acad. Sci. 873851 (1990); Beck, Nucleic Acids Res. 175115
(1989)). Automation of gel electrophoresis based technologies
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demands clear, relatively unambiguous detection systems for
operator-free function (Lehrach, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y. pp39-81 (1989); Larin, Proc. Natl. Acad.
Sci., 884123 (1991)). Sophisticated computational methods can
extract usable data automatically from difficult conditions. A
good example of a fully integrated approach to mapping comes
from the Cohen laboratory which has combined all of these
technological approaches together with "mega-YACs" (Bellanne-
Chantelot, et al., Cell 70L1059 (1992)) to maximally boost
output to a dramatic extent, although with problems inherent in
the fidelity of these YACs (Anderson, Science 2591684 (1993)).
Construction of physical maps for eucaryotic
chromosomes is laborious and difficult, in part because many of
the current methodologies for mapping and sequencing DNA were
originally designed to analyze genes rather than genomes, so
that at present there is a premium on automating procedures such
as PCR and blot hybridizations (Chumakov, Nature 359380 (1992)).
Two techniques have played a fundamental role in the process of
ordering and sizing DNA sequences from eucaryotic chromosomes.
Electrophoretic methods have the advantage of good size resolu-
tion, even for long chains, but require DNA in bulk amounts.
Sources include genomic DNA or YACs (Burke, Science 236806
(1987)). Single molecule techniques, such as fluorescence in-
situ hybridization or (FISH), utilize only a limited number of
chromosomes (Manuelidis, J. Cell. Biol. 95L619-625 (1982)) but
have not yet attained a sizing capability comparable to that of
pulsed electrophoresis. Ideally, one would like to be able to
combine the sizing power of electrophoresis with the intrinsic
loci ordering capability of FISH in order to construct accurate
restriction maps very rapidly.
All considered, the evolution of various physical and
genetic techniques has enabled far more to be accomplished than
expected toward creation of a complete, physical map of whole
chromosomes and the entire human genome (Bellanne-Chantelot, et
al., Cell 70L1059 (1992); Chumakov, et al., Science (1992);
Mandel, et al., Science 258103 (1992)). Despite this progress
the situation can be improved in the following areas.
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For fingerprinting YACs, chromosomal DNA is digested
with several enzymes and then blotted and sometimes hybridized
with several different repetitive sequences (Bellanne-Chantelot,
et al., Cell 70L1059 (1992); Stallings, et al., Proc. Natl.
Acad. Sci. 876218 (1990); Ross, et al., Techniques for the
Analysis of Complex Genomes, Academic Press, Inc., San Diego,
CA, (1992)). Here, electrophoresis is used to size restriction
fragments that are specifically identified by hybridization.
The data density available for such an analysis is relatively
low. For example, it is difficult to discern more than 100
bands in a given land in a typical agarose gel. Additionally,
restriction fragments that are the same size cannot be resolved
from each other and can only be discerned by careful,
differential hybridization. Therefore, the fingerprint does not
report nearly as much information as what would result if an
ordered restriction map were to be made with the same enzyme(s)
or even an accurate histogram of the size population. Such a
histogram can only be obtained from gels by difficult
measurements of band fluorescence intensities.
Gels are time-consuming. It takes time and care to
pour gels and minutes to days to run, and it can take several
days to do Southern analysis, although gels offer the
opportunity for parallel sample analysis and, with multiplexing
techniques (Church, et al., Science 240185 (1988)), this
tremendous ability is probably maximized, sizing results are
often difficult to digitize and to automatically tabulate.
Electrophoretic size resolution for commonly run
agarose gels rarely exceeds mass. Although under limited
conditions greater size resolution can be obtained (Calladine,
Journal of Molecular Biology 221981 (1991)). Greater size
resolution would enable simpler fingerprints with a higher
information content. Although pulsed electrophoresis techniques
can, under certain circumstances, boost size resolution, these
results can be hard to interpret except in very narrow size
ranges. Ultimately, these measured sizes are dependent on size
markers which are limited in range for very large DNA molecules.
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For pulsed electrophoresis, the determined size is frequently
inadequately interpolated between several size markers.
(iv) Usable sensitivity is limited to the subpicogram
range except by exotic techniques (Glazer, et al., Nature 359859
(1992); Quesada, BioTechniques 10616 (1991)). However, now
common phosphor imager systems have improved sensitivity some
and make quantitation easier. The usable sensitivity range will
dictate the type of sample that can be analyzed. For example,
single-copy mammalian genomic hybridizations can be challenging
to a novice. Mapping of end-labeled partial digestion of
genomic DNAs is often not successful because of loss of
attending sensitivity (Smith, et al., Nucleic Acids Res. 32387
(1976)) so that extensive analysis is difficult to do with
genomic DNA samples. This necessitates the reliance on cloned
genomic material, despite their limitations including problems
with uncloneable regions, rearrangements and deletions.
Although YACs enable cloning of such large genomic fragments and
have served as the basis for many mapping approaches, they are
not perfect mapping reagents and must therefore be used with
great caution (Anderson, Science 2591684 (1993); Vollrath, et
al., Science 25852 (1992); Foote, et al., Science 25860 (1992)).
Citation of documents herein is not intended as an
admission that any of the documents cited herein is pertinent
prior art, or an admission that the cited documents are
considered material to the patentabilty of the claims of the
present application. All statements as to the date or
representations as to the contents of these documents are based
on the information available to the applicant and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
SIIbDLARY OF THE INVENTION
The present invention provides methods for physically
characterizing large or small molecules, including polymers and
particles, for determining molecule size, molecular weight, size
distribution, weight distribution and/or enzyme cleavage maps of
a homogenous, heterogenous, or polydisperse or varying sample of
molecules.
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This invention can determine at least one of molecule
size, weight and cleavage or restriction maps using faster and
more efficient methods, which also can provide better
resolution, than methods known in the related art.
This invention also can size one or more particles or
molecules using an extremely sensitive method, e.g., one which
can use an amount of sample, e.g., as small as a few molecules
or a single molecule.
Accurate size information for a polydisperse sample
containing molecules having a wide range of sizes, is also
provided, as well as providing this information more quickly
than by using previously known techniques.
Methods of the present invention involve characteriz-
ing individual molecules, including deformable and non-
deformable molecules, in a polydisperse sample by placing the
molecules in a medium, applying an external force to the
molecules, thereby causing physical changes (particularly
conformational and/or positional changes), and then observing
and measuring these changes. This method is useful for
characterizing molecules of a variety of sizes, including the
smallest molecules which are detected by a suitable microscope
(the microscope optionally may be attached to a spectroscopic
apparatus and thus molecules too small to be visualized may
still be detected), and large polymers, which may be up to
several or many inches in length when stretched to a linear
conformation. Shear sensitive molecules (e.g., large
molecules), which cannot be placed on a microscope slide without
breaking when conventional techniques are used, are measured
according to this invention by collapsing (condensing) the
molecules before they are placed in the medium and then
uncollapsing them after placement in the medium. This invention
is useful for characterizing many types of particles which can
be visualized or detected under a light microscope. Several
non-limiting examples include polysaccharides, polypeptides,
proteins, and nucleic acids (e.g., DNA or RNA).
Deformable molecules are particles or molecules which
have a tendency to change conformation (shape), as well as
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position, when they are subjected to an external force. Non-
deformable particles or molecules tend to have a substantially
stable conformation even when subjected to an external force,
but may undergo changes in position. Deformable molecules are
usually reversibly deformable, e.g., they change conformation
when an external force is applied, and then return to a
configuration comparable to their original shape when
application of the force is terminated.
This invention is particularly useful for measuring
polymer molecules which are folded, coiled and/or supercoiled
and are subject to conformational changes such as stretching,
bending, twisting, contracting, and the like, as well as
positional changes such as rotating, translating and the like.
This invention is particularly useful when an external force is
applied to molecules which are in some type of medium. However,
if a free solution is used, application of an external force may
not be needed to cause the molecules to change conformation or
position.
Molecules which are large enough to be seen using a
microscope are measured by visualization, e.g., by direct
observation of a microscopic image. Particles may,
alternatively, be measured using microscopy combined with any
suitable spectroscopic technique, particularly if the particles
are too small to be imaged (viewed with acceptable resolution).
Several non-limiting examples of useful spectro-scopic
methods include using polarized radiation as generated by a
laser combined with measurement of refractive index or
fluorescence dichroism, or using sensitive video cameras such as
cooled charged coupled devices, silicon intensified target
devices, and micro-channel plate detectors.
Samples containing a mixture of both small and large
molecules, for example, small and large DNA molecules including
chromosomes, are sized rapidly, with each molecule in the sample
being measured simultaneously. The method of this invention
involves measuring conformational and positional changes of
individual, discrete molecules (or other particles), as
contrasted to methods known in the art, which characterize a
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sample in bulk. The method of this invention may be applied to
measure any number of molecules, ranging from a single molecule
to a large number of molecules. If a sample containing a large
number of molecules is measured, the number of molecules which
are observed at one time will depend in part upon the field of
view of the microscope and the extent to which the molecules are
separated from each other. Viewing discrete, individual
molecules, or measuring their role of relaxation after applying
an external force permits complete deconvolution or separation
of measured parameters.
The medium used in this invention is any suitable
material. Preferably the medium will hold relaxed molecules in
a relatively stationary position and yet permit movement of
molecules which are subjected to an external force. However, a
free solution also may be used. For measurements of molecular
movement, a suitable medium is any medium which will permit
different molecules to change conformation and position at
different rates, depending upon their size, and perhaps upon
their chemical composition.
For many uses of this invention, the preferred medium
is a gel or a liquid. Preferably, the medium is anticonvective,
but this is not absolutely necessary. The medium may or may not
be inert. The choice of an appropriate medium will depend in
part upon the size of the molecules which are measured, the
tendency for the molecules to change position and shape, and the
desired precision of the measurements. For example, when large
molecules (or other molecules of similar size) are measured, a
gel with a large pore size is preferably used.
The external force applied to the molecules is any
force which causes the nondeformable or deformable molecules to
undergo changes in conformation or position. For example, the
force may be an electric field, solvent flow field, or a
magnetic field, but is not limited to these types. The force
may vary in direction, duration and intensity. A particularly
useful way to perturb the molecules is by using electrophoresis,
or by site specific enzyme digest, e.g., restriction enzyme
digest of a DNA molecule.
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The types of changes which are measured in this
invention primarily include changes in conformation or shape,
including stretching and relaxation rates, as well as length and
diameter (or radius) measurements, and changes in position,
including changes in orientation and rotation as well as
translation within the medium. Molecules may undergo changes in
conformation or position, or both. Different types of changes
are measured according to various embodiments of the invention.
The techniques for measuring conformational and
positional changes include, but are not necessarily limited to,
microscopy (alone), and microscopy combined with spectroscopy.
Several non-limiting examples of useful spectroscopic techniques
include birefringence, linear or circular dichroism, and
detection of fluorescence intensity.
Molecules which are large enough to be seen under a
microscope can be measured by visualizing (imaging) the molec-
ules. As non-limiting examples, a light microscope or a
scanning/tunneling microscope may be used. While molecules may
be viewed directly, it is useful to link the microscope to a low
light sensitive video camera, connected to a computerized image
processor (e.g., as described herein or as would be suggested to
one skilled in the relevant arts) which records a series of
photographs, even a motion picture, by digitizing or recording
the images which are received. The image processor may itself
comprise a computer, or may be linked to a computer which
processes data based upon the images. Use of a computerized
apparatus enables the movement of each individual molecule to be
measured simultaneously. Furthermore, the relationship of
molecules to one another may be detected, and several different
parameters of a single molecule can be measured simultaneously.
Optionally, the microscope and image processor are
connected to a spectroscopic apparatus. This technique is
particularly useful for molecules which are too small to be
visualized, but is also useful for sizing larger molecules as
well.
In order to transform measurements of change in
conformation and position into size measurements, it is
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generally necessary to generate (or otherwise obtain) data
relating to physical changes of molecules of known size when the
molecules are subject to external forces. "Markers" are
developed by measuring the parameters of molecules with known
values of molecular weight. This information may be input into
a computer or data processor in order to establish a relation
between molecular weight and particular conformational and
positional changes which are measured. Preferably, the markers
are molecules of similar chemical structure to the molecules of
unknown size (e.g., both molecules contain the similar chemical
components), because rates of relaxation, reorientation and
rotation may be dependent upon molecule composition. However,
this may depend upon one or more other variables, e.g., polymer
size, composition, molecular weight, pKa, amino acid or nucleic
acid sequence, etc., and thus it may not always be necessary for
the "markers" to have a composition similar to that of the
molecules of unknown size.
Shear sensitive molecules are molecules which are
subject to breaking when they are placed on a microscope slide
using conventional methods. According to another aspect of this
invention, such molecules may be collapsed into a higher density
conformation before they are placed in a medium, in order to
prevent breakage when the molecules are mounted on a microscope
slide. Once they have been placed in the medium, they can be
uncollapsed and measured by the same methods as the smaller
molecules.
In one embodiment of the present invention,
fluorescently stained, deformable molecules which are coiled,
folded or otherwise configured in a native relaxed, folded or
complexed conformation are placed in a medium and are
temporarily deformed, related, unfolded, separated, cleaved or
stretched by applying an external force. When application of
the force is stopped, the relaxation or reversion time of the
molecules (e.g., the time required for the molecules to return
to their original, native state) is determined by direct
microscopic observation of molecular movement or change, or by
at least one of microscopy and spectroscopy. Alternatively, the
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kinetics of stretching are measured by following the stretching
of the molecule after initiation of the external force. Rate
measurements are calculated in various ways, for example, by
determining an amount of change per unit time. Rates of change
for molecules of unknown size are determined based upon rates of
molecules of known size, such as by interpolation or
extrapolation.
As, e.g., with the viscoelastic measurement technique
known in the art, the relaxation time of molecules in a liquid
according to this embodiment varies as about M'-". In a gel, it
is believed that resolution may be as high as MZ4. This is based
upon known theoretical principles which show that molecules
reptate in gels or confining matrices, and their relaxation time
is much greater in a gel than in a solution (see, e.g.,
DeGennes, P.E., Scaling Concepts in Polymer Physics, Cornell
University Press, N.Y. (1979)).
In a second embodiment, the reorientation time of a
deformable or non-deformable molecule is measured. When
molecules are first subjected to a perturbing force in one
direction, and the direction of the perturbing force is then
changed, for example, by 900 or other transverse angle, such as
10-90 , 20-90 , 30-90 , 40-90 , 50-90 or any range or value
therein, small molecules quickly reorient themselves and start a
new migration along the new path. Larger molecules, on the
other hand, remain substantially immobile until they are
reoriented in the direction of the electric field. Then, they
too begin to move in the new direction. By that time, the
smaller molecules will have moved ahead. Measurements of the
rate at which the position of a molecule changes with respect to
an external force may be measured, for example, by measuring
changes in position (e.g., lateral and/or rotational movement)
per unit time.
In a third embodiment, the rate at which a molecule
rotates is determined when a series of external forces are
applied. This method is particularly applicable to rod-shaped
molecules, such as small DNA molecules, and elongated molecules
which are maintained in a relatively uniform conformation.
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"Rotation time" according to this invention is the amount of
time required for a molecule to undergo a positional rotation of
a particular angular increment, for example, 3600, when a
particular set of external forces are applied.
By periodically switching pulse direction, intensity
and length, molecules are caused to move slightly back and forth
as they are rotated. This facilitates rotation, and is
analogous to the way in which an automobile is manipulated into
or out of a parallel parking space by alternating backward and
forward motion. However, unlike an automobile, a rod-shaped or
coil molecule may bend somewhat as it rotates. A pulsing
routine may also function to keep a deformable molecule in a
generally consistent conformation, in order to provide useful
measurements, e.g., measurements which relate rotation time to
molecular size.
Data for reorientation and/or rotation rates for
molecules of known size may be used to develop a relationship
between reorientation and/or rotation rate and molecular size,
which then may be used to determine the size of various polymer
molecules of similar composition and unknown size, such as those
which are present in a polydisperse sample. Reorientation and
rotation rate may be determined using microscopy (preferably
combined with image processing) to directly observe positional
changes, or by combining microscopy with spectroscopic measure-
ments. Thus, these embodiments are useful not only for mid-
sized and large molecules, but also for molecules that are too
small to be imaged with acceptable resolution.
In yet another embodiment of this invention, the
length of a molecule which has been placed in a medium is
directly measured using microscopy. This technique provides
direct measurement of the molecular size of any number of
molecules. This method generally involves observing the
curvilinear length of deformed molecules which are in a
stretched state, e.g., during the application of an external
force, or soon after termination of a force which has stretched
a molecule. However, this method also may be applied to non-
deformable molecules having an elongated shape, and measurement
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of such molecules does not require application of an external
force before measurements are made. Preferably, this embodiment
uses the same microscopy and imaging equipment as is described
above.
In a fifth embodiment, the diameter (or radius) of
molecules or other molecules suspended in a medium is measured.
Application of a perturbing force is optional, because the
diameter of a deformable molecule is preferably measured when
the molecule is in a relaxed state, and the molecule is
spherical, ellipsoidal or globular in shape. This embodiment
may be used to measure molecules which are deformable or non-
deformable, and involves the use of a light microscope attached
to a computerized imaging device.
These five embodiments may be combined such that some
or all of the above-mentioned parameters are measured simulta-
neously for one or more molecules.
A sixth embodiment of the invention is directed
particularly to sizing very large molecules which tend to break
if they are mounted on a microscope slide using conventional
methods. In brief, this new technique involves collapsing the
molecules before they are placed in the medium, using an agent
which causes them to condense, and then uncollapsing the
molecules after they have been placed in the medium. The
molecules are then sized according to the method of embodiments
one to five. The method for chemically collapsing molecules
also may be used when it is desirable to place a large number of
molecules in a small area, such as in microinjection, even if
the molecules are not large or shear sensitive.
This invention provides a novel technique for mapping
nucleic acid molecules. For example, when a nucleic acid is
placed in a matrix and digested, the fragments are ordered by
the computerized apparatus, and are sized by the methods
described above. Thus, the order of the digests is quickly and
accurately determined.
A further aspect of this invention provides for
sequencing nucleic acid molecules by hybridizing probes to
portions of a molecule. A nucleic acid is placed in a medium,
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to which suitable, desired probes are added. At least one
restriction enzyme may also be added. Reaction is initiated by
an appropriate means, for example, the addition of ATP
(adenosine triphosphate) and/or magnesium ions. After the
probes have hybridized they are detected by the methods
described above, namely, microscopy (alone) or microscopy in
combination with spectroscopy.
Thus, the present invention provides an accurate
method of determining the size of individual molecules and the
weight distribution of a polydisperse sample of molecules.
Another important advantage of this invention over the
techniques of the prior art is that the measurable parameters
for each molecule in a polydisperse sample, not just the largest
molecule, are determined. Additional advantages are that (1)
only one molecule is needed, and the sample may be very small,
e.g., may consist of only one, or only a few molecules (2)
measurements may be based on one representative molecule for
each size in the sample, (3) the technique can be used for very
large molecules (molecules too large to be measured by prior
known methods), (4) data can be processed efficiently by
computer, (5) measurements can be made more rapidly than methods
known in the prior art (e.g. particularly as compared to slow
electrophoresis processes, which may take several weeks), and
(6) measurements are extremely accurate.
The present invention also can provide analysis of
chromosomally sized DNA molecules and associated complexes
utilizing new, ultra-rapid methodologies for determining the
organization and structure of a eukaryote, such as an animal or
human genome. Methodologies are provide which permit high
resolution mapping for multiple individuals in a population of
animals such as humans.
Such methods may involve optical mapping, which is a
nonelectrophoretic approach, to rapidly create high-resolution
ordered maps from chromosomally sized DNA molecules. Optical
mapping produces ordered maps by fluorescently imaging single
DNA molecules during restriction enzyme digestion. The
resulting fragments are then sized by a number of single
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molecule methodologies according to the present invention. To
facilitate mapping of mammalian genomes, optical mapping can be
used to extend the size resolution to map fragments consisting
of a few hundred base pairs, in addition to increase precision
and throughput. To accomplish this, advanced intensity
measurement techniques and sizing methodologies based on
molecular relaxation can be used according to the present
invention, as well as modified chamber designs and fixation
techniques.
Detection Methods for Localization of Sequence
Specific Sequences Including Hybridization to Single DNA
Molecules. RecA protein-mediated hybridization approaches are
also provided by the present invention, to precisely map
sequences of large DNA by methods which may include optical
mapping. Large target molecules can be imaged, localized and
quantitated at specific sites via the visible gaps produced in
the molecules at the hybridization site. Such sequence
localization techniques can be combined in the present invention
with sizing methodologies and sample handling techniques to
improve throughput and versatility. Another method of the
prese,nt invention is direct imaging of hybridization sites; this
is based on conjugating RecA oligonucleotide filaments to
different types of optically detectable tags allowing direct
visualization of hybridization sites. Energy transfer
techniques can also be used to enhance the specificity and
effectiveness of tagged RecA-mediated hybridization as detected
by imaging.
Increasing Throughput in mapping using Genomic DNA or
YACs A number of single molecule methodologies can be combined
in the present invention to dramatically increase the production
of maps from YACs or genomic DNA. High throughput, flow-based
optical mapping systems provided by this invention e.g., by
producing high resolution, ordered restriction maps. RecA-
assisted restriction endonuclease cleavage (RARE), according to
this invention can selectively dissect genomes into large
fragments. RARE and related techniques provide optical mapping
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approaches to rapidly map large genomic regions, without the
need for cloning or sequencing.
Such optical mapping methodologies provide analysis
methods for complex mammalian genomes, e.g., by applying sizing
methodologies to raise the level of molecular size
discrimination. These applications are facilitated by the
discovery that relaxation phenomena of polymer molecules, e.g.
DNA, demonstrate a remarkably high degree of size dependency.
High throughput interfaces of methodologies of the present
invention are also provided. The combination of high resolution
and high throughput are preferably used with methods of this
invention to provide high resolution maps of entire populations
of a given species or animal subgroup. The value of cataloging
high resolution maps of individuals for global genome
comparisons is enormous, e.g., genetic analysis, and use in
clinical settings for detecting complex heritable disorders,
resulting from multi-genic determinants.
These and other advantages will become readily
apparent from the detailed description which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic drawing of an electrophoretic
microscopy chamber which is specifically adapted to fluorescence
microscopy studies.
Figure 2 is a partly schematic and partly block
diagram showing an interconnection of exemplary chamber
electrodes in an electrophoresis chamber which may be used in
the present invention.
Figure 3A-B is a schematic illustration of the
instrumentation used in the microscopic study of DNA molecules
in a medium according to this invention, and a more detailed
diagram showing the instrumentation for measuring birefringence.
Figure 4A-I shows the DNA molecular conformational and
positional changes when G bacteriophage molecules are subject to
two sequential electric fields in different directions.
Figure 5A-J shows the DNA molecular conformational and
positional changes during relaxation of G bacteriophage DNA
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molecules after electrophoresis for 600 seconds, as revealed by
the fluorescence microscopy experiments described in Example 4.
Figure 6 shows optical mapping, where DNA molecules
and restriction enzyme are dissolved in molten agarose without
magnesium ions. The DNA molecules are elongated by the flow
generated when the mixture is sandwiched between a slide and
coverslip. Stretched molecules are fixed in place by agarose
gelation. Magnesium ion diffusion into the gel triggers
digestion and cleavage sites appear as growing gaps as the
molecular fragments relax.
Figure 7A-D shows histograms of optical mapping, with
Not 1 cut frequencies showing variation with molecule size and
number of cut sites. Cutting frequencies were scored by
counting cuts in molecules present in microscope fields
(containing typically 3-5 molecules). Because about half the
fields showed no cutting and were not scored, this
underestimates the number of uncut molecules. The expected
number of cut sites and chromosome sizes, from (14) 7(A) Ch. 1,
1(240 kb), 7(B) Ch. V and VIII, 3 and 2 (595 kb), 7(C) Ch. XI, 2
(675 kb), and 7(D) Ch. XIII and XVI, 1 (950 and 975 kb).
Chromosome pairs V and VIII, and XIII and XVI coexist in the
same mount.
Figure BA-H shows some restriction fragment relaxation
modes for a singly cleaved, gel-fixed, elongated molecule.
Horizontal arrows indicate direction of relaxation. Relaxation
modes illustrated 8A fixed molecule before cleavage, 8B-E
possible relaxation modes producing detectably cleaved
molecules, and 8F-H relaxation modes producing undetectably
cleaved molecules.
Figure 9 shows as graphic represent depicting possible
relaxation events to form pools of segments or "balls" at coil
ends. Agarose gel is illustrated as a series of pegs with free
spaces available for molecules. Gel pegs might intersect the
embedded DNA molecule during gelation and possibly entrap it.
The coil segments positioned in the pool region comprise a
relaxed sub-coil region and have higher entropy than the coil
stretched out between them. These pools may act as molecular
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rivets in some circumst,apces, particularly if the segment pool
mass approaches that ofthe intervening coil.
Figure l0A-B shows optical mapping sizing results for
Not I endonuclease restriction fragments from S. cerevisiae
chromosomes I, V, VIII, XI, XIII, and XVI calculated as
described, plotted against published results. The diagonal line
is for reference. Typical fragment images are shown in this
figure. See example 13. The inset shows the estimate of
population standard deviation (kb). Error bars represent 90%
confidence (7) on means (main graph) or standard deviation
(inset). 10A shows the relative intensity determination of
fragment sizes. lOB shows the relative apparent length
determination of fragment sizes.
Figure 11 shows a scatter plot of normalized absolute
intensity vs. apparent length. Absolute intensities from
individual images were calculated (6) and plotted against
apparent length over a time interval typically used in optical
mapping (10-15 minutes). For each sample, the initial intensity
was found by averaging absolute intensity values from groups of
5 adjacent images and taking the largest value. The values from
several samples were normalized by dividing values from each
image by the initial intensity for the sample. (A) chromosome I
120kb Not I fragment, 7 samples. (B) chromosome XI 285kb Not I
fragment, 4 samples. (C) chromosome XI 360kb Not I fragment, 4
samples.
Figure 12 is a graphical comparison of Not I
endonuclease restriction maps of optical mapping results of S.
cerevisiae chromosomal DNA molecules with published restriction
maps (L&0). Maps were constructed from length (Len), intensity
(Int) or a combination of both (Com). Bar lengths for the
optical mapping data are proportional to the means plotted in
Fig. 10A-B, and typical images are shown in Fig. 13A-F.
Figure 13A-F shows typical fluorescence microscopy
images of S. cerevisiae chromosomal DNA molecules stained with
DAPI and embedded in agarose gel during Not I restriction
endonuclease cleavage. Chromosomal DNA molecules were prepared
and fixed as described in example 13 and cited references.
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Images were background corrected using a smoothed and attenuated
background image, smoothed, and stretched, using 16-bit
precision. Images show Not I restriction digestion evolution,
with arrows highlighting cut sites. Intervals are timed after
addition of Mg+2. 13(A) Ch. I (240kb), 20 and 60 sec; 13(B) Ch.
XI (675kb), 500, 880 and 1160 sec; 13(C) Ch. V (595kb), 200,
240, 520 sec; 13(D) Ch. VIII (595kb), 440, 1220 and 1360 sec;
13(E) Ch. XIII (950kb), 100 and 560 sec; 13(F) Ch. XVI (975kb),
460 and 560 sec. Bars, 5 m. A 100x objective was used to
image results in panels (13A-D) and a 63x objective was used for
panels (13E and F).
Figure 14 shows optical mapping results from Rsr II
and Asc I endonuclease restriction digest of S. cerevisiae
chromosomes III and XI. Maps were constructed from fully cut
length (Len) or intensity (Int) data, and refined using partial
cut length. Bar lengths are proportional to the calculated
means, and typical images are shown in Fig. 15. Number of cuts
was determined as in Fig. 7.
Figure 15A-C shows fluorescence microscopy images of
S. cerevisiae chromosomal DNA molecules stained with DAPI and
embedded in agarose gel during Rsr II or Asc I restriction
endonuclease cleavage. Chromosomal DNA molecules were digested
and analyzed as in Fig. 13. Images show restriction digestion
evolution, with arrows highlighting cut sites. 15(A) Ch. III,
Rsr II, 1100 and 1820 sec; 15(B) Ch. XI, Rsr II, 20, 600, 920,
1060 sec; 15(C) Ch. XI, Asc I, 1160, 1500, 1780, 1940 sec. An
isoschizomer to Rsr II, Csp I, was also used and gave identical
results. Bar, 5 m.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods for
characterizing physical and/or chemical properties of labeled
and non-labeled molecules in a medium. Such properties may
include but are not limited to, at least one of length, size,
restriction maps, weight, mass, sequence, conformational or
structural change, pKa change, distribution, viscosity, rates of
relaxation, reorientation and rotation or other property of
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molecules subject to an external force, during or after the
molecules are subjected to an external force.
Among other applications, these measurements may be
used to determine molecule size. The invention is suited to
size polymer molecules in a polydisperse sample (e.g., a sample
containing molecules of varying size), when the molecules have
been placed in some type of medium, and is useful to measure
very large molecules, such as large nucleic acid molecules,
which are subject to breakage when placed on a microscope slide
using conventional methods. In several embodiments, molecules
are measured either during or after they are deformed or
repositioned by an external force. The method of this invention
is particularly suitable for examination of nucleic acids and
other polymers which are coiled, or possibly even supercoiled,
when they are in a relaxed (unperturbed) conformation.
Methods for determining the molecular weight distribu-
tion of a polydisperse sample of molecules are useful in a
variety of different fields. For example, in polymer chemistry,
the properties of an oligomer are often dependent upon its
molecular weight distribution. When a particular substance is
found to exhibit favorable properties and the exact composition
of the oligomer is not known, an analysis of the molecular
weight distribution of the polymer is used for purposes of
identification. In molecular biology, the molecular weight
distribution of a polydisperse sample, such as a sample of DNA
restriction enzyme digests, provides valuable information about
the organization of the DNA. This information may be used to
produce chromosome maps and extensive molecular genetics
characterizations.
Molecules are placed in a medium, such as a solvent,
powder, glass or gel. Other suitable mediums also may be used.
The medium containing the molecules may be placed on a
microscopic slide, or may be positioned in some other manner so
as to permit the molecules to be viewed under the microscope.
The suitability of a particular medium may depend in part upon
the measurement technique to be used. Measurement techniques
requiring perturbation necessitate the use of a medium in which
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deformable molecules are capable of changing conformation or
position. When direct observations are made under a light
microscope, a suitable medium also allows the molecules to be
viewed clearly. When microscopy is combined with birefringence
measurements, a preferred medium prevents convection and enhance
the size dependency of the observed signal. Additionally, the
medium itself preferably is relatively free of significant
birefringence during experimental conditions.
Characterization of molecules based upon measurements
of fluorescence intensity may be greatly enhanced using a matrix
(e.g., a medium which partially confines molecule movement) as
the medium. For the method of this invention, the medium is
preferably a solution or gel. Agarose and polyacrylamide gels
are particularly well suited for use in this invention.
Examples of suitable solvents include glycerol/water,
polydextran/water, and organic solvents. However, these
examples are not to be construed as limiting the scope of the
invention.
In a preferred embodiment, agarose, a polysaccharide
derived from agar having an average molecular weight of approxi-
mately 100,000 daltons, is dissolved in an aqueous buffer
(typically a i% solution) and allowed to cool, forming a rigid
gel, similar to gelatin (as found in Jello). The gel matrix
consists of a three-dimensional network of agarose polymer
chains annealed to each other through hydrogen bonding. Heating
the agarose gel will send it back into a fluid state, so that
the gel is said to be reversible, just like gelatin. The
important feature of an agarose gel is its extraordinarily large
average pore size. Although the pore voids in agarose gel are
presently not completely characterized, they are thought to be
approximately 0.3 microns wide, and also contain smaller voids.
Due to its large pore size and inertness, agarose is used in DNA
gel electrophoresis, because it allows DNA molecules to stretch
and move in a gel.
If a flourescence microscope is to be used (or if
fluorescence intensity is to be measured by other means), the
molecules generally are stained. Staining procedures are well
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known in the art. Useful stains or chromophore in this invention
include, but are not limited to, ethidium bromide and 4',6-
diamidino-2-phenyl-indole, dihydrochloride (DAPI). Most types
of molecules are stained at some time before they are imaged,
and may be stained before or after they are placed in a medium.
When a deformable molecule is placed in a medium and
mounted on a slide, there are several possible ways to perturb
the molecule. Several non-limiting methods are as follows. (1)
A molecule is perturbed by the application of an electrical
field, which moves a charged molecule such as DNA through a
matrix, distorting the coil conformation much as cookie dough
distorts as it moves through a forming machine. This is the
phenomenon involved in gel electrophoresis. (2) A flow field is
created in the liquid agarose/DNA (or polymer of choice) and the
molecule-containing liquid is then gelled quickly with low
temperature quenching, fast enough to prevent any significant
coil relaxation. This method is useful whether or not the
molecule is charged. (3) Using the dielectrophoresis effect,
uncharged molecules with field gradients (electric fields which
change strength with position) are moved by distorting molecular
electron clouds, thus inducing attractable dipoles. It is
believed that dielectrophoresis could be used in a matrix such
as agarose.
One of the advantages in using electrophoresis is that
DNA molecules are distinguished from other molecules which may
be present in the DNA-containing medium because uncharged
molecules do not move in response to application of an electric
field. Another possible way to distinguish DNA molecules from
others is to multiply the DNA.
The extent to which a molecule is to be preturbed,
e.g., subjected to changes in conformation and/or position,
before measurement, may vary. For example, useful data on
molecular relaxation is obtained even when a coiled molecule is
perturbed such that it is only partially uncoiled.
As mentioned above, one of the preferred methods for
perturbing molecules according to this invention involves
electrophoresis. Any electrophoresis method suitable for use
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with a microscope may be used according to the present invention
to perturb the molecules. Electrophoresis may optionally serve
another function in this invention. When a sample size is too
large or complex to be viewed under a microscope all at once,
molecules may be separated into sub-samples which then are
imaged separately.
Electrophoresis of molecules for viewing and measuring
purposes may optionally use a chamber which is suitable for use
in pulsed field electrophoresis, as described in U.S. Patent No.
4,695,548, or for pulsed oriented electrophoresis, which is
described below. These techniques are particularly useful in
the event it is desirable to measure reorientation and/or
rotation times, because of the ability to control field angles.
Pulsed Oriented Electrophoresis (POE), which was
developed by'the present inventor and is the subject of co-
pending application number 07/244,897, improves separation of
polydisperse polymer molecules in a sample by using short
electric pulses to create and vary field angles, with the
effective field angle being defined by the vector sum of a
series of pulses which may vary in duration, intensity and
direction. Pulse times and pulse intensities are modulated to
effect separation. POE is also useful for creating effective
field angles during imaging. The needed instrumentation is
readily adapted to the microscope.
An exemplary laboratory instrument for POE is illus-
trated in Figure 1 and a schematic view is shown in Figure 2.
The instrument exemplified in Figure 1 is similar to a
miniature version of that described in U.S. Patent No.
4,473,452, but differs in that the POE instrument has two sets
of diodes 34 which enable bipolar operation of the discrete
electrode array. The diodes 34 can be replaced by a multiganged
relay (not shown) to provide similar electrical isolation.
However, it is best to use the diodes 34 when very fast (less
than 1 second) pulsing is needed.
As depicted in Figs. 1 and 2, the miniature
electrophoresis chamber 50 used in this invention measures about
the size of a standard coverslip. It has electrodes 421, which
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are connected to diodes 34 (Fig. 2). In order to generate the
desired electric fields, platinum electrodes 42' are intercon-
nected as shown in Fig. 2. In particular, d-c power supply 28
supplies d-c power to relays 30, which are controlled by a
computer 32 to connect selected outputs to the d-c power from
power supply 28. Computer 32 also controls d-c power supply 28
so that the potential of the power supply can be varied.
Outputs to relays 30 are connected to electrodes 42' through
respective diodes 34 for each electrode.
As shown in Fig. 1, the miniature POE apparatus has a
holder 52, which fits on a microscope stage. A slide 54, which
holds an agarose gel, is placed into the holder and the elec-
trodes 42 make electrical contact with the slide/gel/cover-slip
sandwich placing drops of 30% glycerol-agarose at the agarose
electrical connecting wicks 44. The glycerol prevents drying
out of the gel. The electrical connector 46, which is part of
the holder 52, provides a link to the bipolar diodes 34 and
pulsing instrumentation shown in Fig. 2.
As in the case of the instrument described in U.S.
Patent No. 4,473,452, the presently exemplified instrument
generates electrical fields which are orthogonal to each other,
which alternate between high and low intensities out of phase
with each other according to the chosen pulsing routine as
described below and which translate the molecules undergoing
separation incrementally through the gel matrix in an overall
direction transverse to the respective directions of the
generated electrical fields. Due to the novel bipolar nature of
the electrode design, it is possible to change polarities,
simultaneously if desired, in addition to alternating high and
low intensities without any significant electrode induced field
distortions.
The determination of effective field angle by a
pulsing routine rather than by placement of an electrode array
permits molecular orientations (and separations) that would
otherwise be difficult. As described in Example 4 below, POE
has been used in DNA imaging experiments. The electrophoresis
apparatus pictured in Figs. 1 and 2 and used in Example 4 may be
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preferred over that of U.S. Patent No. 4,695,548 because varying
the field angle by moving electrodes as taught by conventional
pulsed field electrophoresis is not practical due to microscope
stage physical constraints. However, use of a POE device is not
necessary to practice this invention when the molecules can be
sufficiently perturbed by other means. Conventional
electrophoresis using an apparatus which is about the size of a
microscope slide is another preferred method for perturbing
charged molecules.
In a preferred embodiment of this invention, a small
electrophoresis chamber containing polymer molecules is placed
upon a microscope slide and the polymer molecules are viewed
under a light microscope. As depicted in Fig. 3(a), imaging of
single molecules is accomplished with an epifluorescence
microscope 62 (excitation light comes from above the sample as
from laser 60) coupled to a low light level sensitive video
camera 64 which is connected to an image processor 66, which in
turn is connected to a video monitor 68. The use of
epifluorescence microscopy here in this invention is an
extension of the methodology first developed by Yanigida et al,
in Application of Fluorescence in the Biomedical Sciences, (eds.
Taylor, D.L. et al), 321-345 (Alan R. Liss, Inc., New York,
1986). A high powered oil immersion objective 58 is preferably
used in the microscope. The key requirement is to use
objectives with a high numerical aperture which gathers light
effectively. A silicon intensified target (SIT) camera or a
micro-channel plate detector is used to boost the light
sensitivity to the point of counting photons. The image
processor is a computer dedicated to digitizing, processing and
storing images from the video camera. This type of image
processor is known in the art. Images can be radically enhanced
to bring up contrast, provide pseudocolor representation of grey
levels (colors can be assigned to enhance images on an arbitrary
basis, not unlike what is done with "colorizing" old black and
white movies), and provide feature analysis which might include
counting objects in the field of view. It is possible to view
single DNA molecules stained with an appropriate chromophore,
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such as 4',6-diamidino-2-phenylindole dihydrochloride (DAPI),
using epifluorescence microcopy.
The smallest size determination of this visualization
technique is limited by microscope resolution, which, at this
time, is approximately 0.1 microns or approximately 300 nucleo-
tide bases in a DNA molecule. This length corresponds to the
length of a small bacterial gene, however some DNA molecules are
up to several inches in length, as is a human chromosome. Since
it is possible to view several molecules in a sample simulta-
neously, it is also possible to measure sizes of many discrete
molecules simultaneously.
An alternate way to size molecules according to his
invention involves measuring molecules spectroscopically. This
technique, when combined with microscopy, is particularly useful
to size molecules which are too small to be imaged with
satisfactory resolution and is described in detail in example 6.
It also may be used for molecules of medium or large sizes.
Resolution using these techniques is simply limited by sig-
nal/noise as determined by photon counting.
The size parameters which are measured according to
the preferred embodiments of this invention include relaxation
or stretching rates of a perturbed molecule, reorientation rate
and/or rotation rate of a particle subject to perturbing forces
in different direction, the curvilinear length of a perturbed
molecule, and the diameter of a spherical, ellipsoidal or
globular molecule. Each embodiment is based upon a mathematical
relationship between the parameter which is measured and
molecular size.
A first preferred embodiment involves measurement of
the time required for a perturbed application of an external
force is terminated. The measurement of relaxation kinetics is
described in Examples 1-4. This embodiment is based in part
upon principles which mathematically relate relaxation time and
molecular size.
An important advantage of measuring fragment sizes
using relaxation rather than by other methods, such as measuring
curvilinear length, is that the DNA molecules does not need to
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be totally stretched out in order to obtain an accurate
measurement. The measured relaxation time is independent of the
degree of coil extension. This has been clearly shown for
measuring DNA relaxation times using the viscoelastic technique
(Massa, D. J., Biopolymers 121071-1081 (1973).
A second preferred embodiment involves measurement of
the reorientation time of a molecule subject to at least one
external force, for example, sequential electric fields in
different directions. This is described below in Example 6 and
is shown in Figs. 4A and 5J. The principles upon which
reorientation rate is based have been studied by the inventor
using fluorescence microscopy/image processing. Using the
process as described below in the Examples, it has been deter-
mined that during pulsed field electrophoresis, the blob train
of a DNA molecule orients with the applied electric field in a
very complicated manner and during this process, electrophoretic
mobility is retarded until alignment is complete, e.g., until
the molecule is aligned with the applied field. Upon field
direction change, the blob train moves in several new directions
simultaneously (i.e., the blobs appear to be moving somewhat
independently). Eventually, some part of the blob train
dominates in reorienting with the applied field and pulls the
rest of the blobs along its created path through the gel. The
time necessary for complete blob train alignment varies directly
with size; i.e., a 10 mb (1 mb = 1,000 kb) molecule requires one
hour to reorient, while a 10 kb molecule requires only ten
seconds, using similar field strengths. The phenomenon is
illustrated in Fig. 4. Reorientation is measured in various
ways, including by light microscopy and by microscopy combined
with spectroscopic methods.
A third preferred embodiment of this invention
involves measurement of the rotation time of a molecule subject
to sequential electric fields in different directions. In one
sense, rotation of a molecules requires a series of incremental
reorientation steps, each of which causes the molecule to rotate
further in the same direction, until the molecule has undergone
a rotation of a specified angular increment, for example, 360 .
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This embodiment is particularly well suited to characterize
stiff, rod-like molecules, such as small DNA molecules, which do
not significantly change conformation upon application of an
external force. However, large molecules also may be sized by
this method if the conformation of the molecules is kept fairly
constant, preferably in a rod-like or elongated conformation.
This is accomplished by applying a pulsing routine which is
appropriate to the size, shape and perhaps also the composition
of the molecule. As a non-limiting example, molecules are
rotated in the presence of sinusoidally varying electrical
fields applied at 90 to each other. Stiff, rod-shaped
molecules or stretched molecules are rotated about the long or
short axis. Rotation about the long axis has the greatest
molecular weight dependence, with rotation diffusion varying as
about M3. Rotational motion of a rod-shaped molecule immersed
in a gel or any other confining may be difficult if an attempt
is made to simply rotate the molecule as a boat propeller
rotates in water. When a gel is used, the matrix affects
rotation of the molecule much as seaweed affects the rotation of
a boat propeller. Thus, a pulsing routine is applied which also
provides back and forth motion of the molecule, thereby
facilitating rotation.
The pulsing routine may be defined by an algorithm.
Generally speaking, the algorithm may depend on variables such
as angle increment, time, electric field intensity, etc., and
these may in turn be a function of different variables. Thus,
the types of usable algorithms are numerous.
A preferred pulsing routine for this invention may
be defined as follows
EI (t) = E (t, O;) (icos0;+] sin0;) (Ot)
E2 (t) = E (t,6;) (icos(6;+7r)+jsin(9;+7r)) (Ot)
P; = K1 * E1 (t) , K2 * Ez (t) , K, * Ei, (t)
wherein
El(t) and E2(t) are electric field vectors multiplied by
time (volt.sec/cm);
E(t,6;) is the electric field intensity in volt/cm;
i and j are unit vectors;
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6; is the field angle, in radians or degrees, with
i = 1-n, where n/E*6;/i=1 = 27r or 3600 for a complete rotation;
Ot is pulse length, in seconds;
t is time in seconds;
kl and k2 are the number of successive identical
pulses; and
P is a pulsing routine, which may be repeated.
Using the above routine, a molecule to which
appropriate pulses are applied rotates about (6;+, - 6;) radians
or degrees when each set of pulses P are initiated. Also, the
molecule is translated (moves laterally) in the directions of
E(t) and -E(t), thereby facilitating rotation.
In the above equation, Ot is a constant, however, this
need not be the case. E may be a function of any variable or
set of variables. For example, E may be a function of total
elapsed time and/or angle increment. Also, the sum of all the
angular increments need not be 360 , and may be any number of
partial or total rotations which provide measurements of
sufficient accuracy.
A specific set of conditions for measuring the
rotation rate of molecules are set forth in Example 7.
According to a fourth preferred embodiment of this
present invention, a useful way to measure the size of molecules
such as polymer molecules is to visualize them and measure their
curvilinear length (equivalent to measuring the length of a
rope) directly~using a light microscope. It is shown in Example
4 below, and in Figures 4 and 5, that fluorescence microscopy
can image single polymer molecules stained with an appropriate
chromophore. Incredibly, even though the polymer diameter
dimensions may only be just 20 angstroms, single molecules are
easily visualized. If the molecule is stretched out and a
computerized imaging apparatus is used to measure the length of
the visualized molecule, the size dependence of the measurements
varies as about Ml. Measurements of length are particularly
useful in sizing and ordering DNA fragments, such as restriction
digests, as described in detail in Example 10.
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A fifth preferred embodiment involves measuring the
diameter of a relaxed molecule. Measurements of molecular
diameter are made according to the same procedure of staining
molecules, placing the molecules in a medium, etc. as the
curvilinear length measurements. However, it is not necessary
to perturb the molecules before measurement. Instead, the
molecules are measured when they are in a relaxed state, having
a spherical or elongated elliptical shape. Because the volume
of a sphere is proportional to R3 where R=radius, and the volume
of an ellipsoid is proportioned to ab2 where a is the radius of
the major axis, and b is the radius of the shorter axis,
resolution for this technique varies as about M33. Molecules
measured by this technique do not need to be deformable. This
technique can be used for all sizes of DNA molecules and is
useful for sizing large DNA molecules, which are now
successfully be mounted on a microscope slide, as well as for
sizing densely packed molecules.
Large molecules, such as large DNA moiecules, are
difficult to mount on a microscope slide without causing
breakage, and the present invention addresses this problem using
a novel technique, which is a further aspect of this invention.
A typical human chromosome may contain a single DNA molecule
stretching inches in length. Nature provides a clever packaging
scheme to fit approximately six feet of DNA into a cell
measuring only a few microns in diameter. However, these large
DNA molecules are very sensitive to breakage. For example,
solutions of large DNA molecules cannot be poured, pipetted, or
stirred without breaking molecules. Thus, working with large
DNA molecules can be very difficult. Some years ago the
inventor developed a gel based method of preparing large DNA
molecules without breakage that also permitted biochemistry
using intact molecules, (see U.S. patent No. 4,695,548). The
procedure is called the insert method and works as follows.
Cells are washed and mixed with low gelling temperature agarose
kept at 37 C. The cell-agarose mixture is pipetted into a mold
(to produce small blocks to fit into the wells of a slab gel)
and allowed to gel. The resulting blocks or "inserts" as they
34
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~D
are named are then placed into a lysis solution containing EDTA,
protease and detergent. The lysis solution diffuses into the
insert, lyses the cells and renders intact naked DNA molecules
stripped of their associated proteins. The DNA molecules do not
diffuse out of the because very large coils are generally unable
to diffuse.
The present invention provides the discovery that DNA
molecules up to at least one megabase (1 megabase = 1 million
bases = 660x106 daltons) when suspended in liquid agarose are
protected against shear when mounted on a microscope slide.
However for molecules larger than 2 or 3 megabases or for
situations where the integrity of 100t of the molecules must be
ensured, this procedure is not effective.
A sixth embodiment of the invention remedies the
above-mentioned problem involved in placing molecules larger
than 1 megabase on a microscope slide. The inventor developed a
protocol using a condensation agent to collapse gel bound DNA
(as obtained from inserts) into small shear resistant balls,
that can be unfolded once mounted, with the addition of an ionic
compound, for example, a salt such as sodium chloride or
magnesium chloride. Preferably, the condensation agent is
spermine. The spermine protocol, which is described further in
Example 10, permits the mounting of DNA molecules of even the
largest known DNA molecules, and feasibly even larger molecules,
without any detectable shear mediated breakage. While the use
of spermine is preferred, other suitable materials for collaps-
ing the molecule include any material which can cause a particu-
lar molecule to collapse, e.g., any condensation agent which
causes molecules to preferentially solvate themselves. Examples
of such materials include, but are not limited to spermidine,
alcohol and hexamine cobalt.
A seventh preferred embodiment of the invention
relates to a specific application of the above embodiments of
this invention to map DNA molecules using restriction enzymes.
The previously known method for constructing a restriction map
is to incubate DNA with a restriction enzyme and size separate
the resulting fragments using conventional gel electrophoresis
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or pulsed electrophoresis. Size separation provides information
on the number and size of the fragments but no information on
the relative location of fragments or cutting sites on the uncut
DNA molecule. By using the microscope to image molecules
undergoing digestion by restriction enzymes, (1) size resolution
is accurately determined by measuring relaxation kinetics, (2)
positioning of fragments relative to each other can be deter-
mined, and (3) only one molecule needs to be digested (however,
many molecules can be image processed in parallel). One
limitation is space on the sample holder.
In brief, restriction mapping using the microscope
involves mounting large gel embedded DNA molecules on a micro-
scope slide, stretching them to some extent (it is not necessary
that the molecules be completely stretched), and then inducing
cleavage. The fragment positions are noted and their sizes are
determined using the methods outlined in embodiments one to
four, using visualization or spectroscopy. A preferred
embodiment of this aspect of the invention is described in
detail in Example 11.
The first step in map construction is to determine the
number of cleavage sites within a molecule by examining
histograms of cuts per molecule and corresponding cleavage
patterns. Because the rates of enzymatic cleavage by different
restriction enzymes are variable, careful adjustment of the
timing is critical. Cleavage preferably occurs only after
molecular fixation was complete because premature reactions
would disrupt attempts to order fragments. As a non-limiting
example, this timing problem can be solved by this invention by
premixing the agarose-DNA solution with restriction enzyme, at
37 C and triggering the reaction by diffusing Mg2+ into the
viewing field, without disturbing the gel. All possible
cleavage sites did not appear simultaneously; instead, cuts
usually appeared within 5 min. of each other. A typical mounted
sample may contain approximately 3 to 5 molecules within a
single viewing field, and overall roughly 50 to 95 s of them
showed evidence of one or more cuts.
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ls c
The next step can be used to determine the size of the
resulting restriction fragments. For this purpose there are
developed two complementary approaches according to this
invention, one based on relative fragment fluorescence intensity
and the second on apparent relative length measurements.
Microscope-based intensity measurements are difficult
to perform because of their dependence on many variables,
including camera control and illumination intensity. By
calculating the relative intensity of two fragments (from the
same parental molecule), one of the fragments serves as an
internal intensity reference for the other. Relative
intensities are converted to kilobases by multiplying by the
known or independently determined chromosome size. Figure 10A
shows the sizes determined for a series of yeast chromosome Not
I restriction fragments measured optically and plotted against
published values derived from electrophoresis-based
measurements. Points close to the diagonal line are in good
agreement. Excluding the two short fragments less than 60 kb
and the low-resolution 8-bit chromosome 5 and 8 data, the pooled
SD was 36 kb (Fig. 10A, inset). The average of the coefficients
of variation was 16%, which is comparable to routine pulsed
electrophoresis size determinations. The correlation with
published results is excellent: the average of the relative
errors is 5t, whereas the published errors average 4t. Due in
part to the intensity normalization procedure, the precision
becomes lower for very small fragments, and size agreement is
poor for the measurements of the 30-and 55- kb DNAs.
Fluorescence intensity measurements report a size of these
fragments almost twice that of the above values.
One test of the validity of relative fluorescence
intensity measurements is to monitor the constancy of fragment
intensities over a usable range of molecular relaxation
conditions. This requirement is most critically tested when
restriction fragments differ greatly in size. Intensities are
discovered to remain relatively constant over a wide size range
despite a three- to four-fold change in measured molecular
length. This beneficial effect can be attributed in part to the
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mild fixation conditions, so that Brownian motion can vibrate
the elongated coil along the z-axis; this motion is clearly
observed on the live video monitor as digestion proceeds. By
averaging frames over a 1-5 interval, most of the DNA which is
observable moves through the focal plane and within the gel
pores.
For measurement of the relative apparent length, each
of the gel-embedded restriction fragments is assumed to have
equal coil density, on the average. Relative apparent lengths
can be converted to kilobases by multiplying by the chromosome
size. Then, the apparent lengths of restriction fragments can
be averaged obtaining accurate sizes from as few as four
molecules. Relative determinations of apparent length can be
standardized against the same set of restriction fragments as in
the fluorescence intensity measurements, and these results (Fig.
lOB) show a similar average relative error of 16% (excluding the
30- and 90- kb fragments). The pooled SD was 47 kb (Fig. lOB,
inset): the average of the coefficients of variation was 29%-,
in these non-limiting examples.
Length measurements can be used to evaluate fragments
that are out of focus, e.g., when out of focus images distort
intensity-based measurements. Additionally, size determinations
of small fragments are better by length than by intensity.
Single Molecule Sizing Methodologies. Optical mapping
is dependent on methodologies for sizing single molecules and
construction of restriction maps of higher resolution and
precision can be provided by such methods of the present
invention. As non-limiting examples, five single molecule
sizing methods are provided, including apparent relative
length, fluorescence intensity ratio, relaxation in a gel,
baseline, and OCM (optical contour mapping). These can be
classified into two groups: techniques that require molecular
perturbation or external force and those that require none.
Non-perturbation sizing techniques such as
measurements of fluorescence intensity of single molecules and
apparent length measurements are convenient to use because they
do not require a sophisticated microscope mounted chamber and
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attending controlling electronics. Additionally, non-perturbing
sizing techniques are also well suited for parallel
measurements. Despite some of these advantages for the non-
perturbing class, the perturbation-based sizing techniques of
the present invention have great utility for characterizing
polymer molecules and mapping chromosomes, as provide higher
precision and resolution.
Figure 12 illustrates three types of ordered
restriction maps produced by optical mapping of the present
invention as compared with published restriction maps.
Additionally, Fig. 13A-F, shows selected corresponding processed
fluorescence micrographs of different yeast chromosomal DNA
molecules digested with Not I. These images clearly show
progressive digestion by the appearance of growing gaps in the
fixed molecules. From such data, the order of fragments can be
determined by inspection of time-lapse images obtained every
time interval, e.g., 0.07-200s, or any range or value therein,
e.g., 1-30s. Because observed molecules tend to move and can
sometimes be confused with other molecules, inspection of a
"cutting sequence" or "cutting movie" simplifies deconvolution
of molecule-molecule interactions. Agreement is expected to be,
and has been found to be excellent, between the optical (length
or intensity) and the elctrophoresis-based maps. The third type
of restriction map (e.g., Com. Fig. 12) combines length- and
intensity-derived data; small restriction fragments (100-20, or
any range or value therein, e.g.<60 kb) can be sized by length,
whereas intensity measurements can provide the remaining
fragment sizes needed to complete the maps.
Fig. 13A shows a non-limiting example of images of a
relatively small yeast chromosome (240 kb) that was elongated
and fixed to roughly one-third of its contour length. Because
chromosomal DNA molecules less than about (e.g., 200-500, or any
range or value therein) relax quickly in molten agarose, trapped
in an extended form at lower temperatures to hasten gelation.
Note that molecular relaxation processes can produce a gap and
form balls at the cut fragment ends, whereas the parental
39
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molecule ends remain essentially fixed. Molecules in this size
range can form disproportionately large balls at parental
molecule ends. Fragment relaxation motions at cut sites can be
observed to re-trace the original gel pores occupied by uncut
molecules as predicted by polymer reptational theory (Figs. 13A-
F, 14 and 15). These molecular characteristics can be conserved
regardless of molecular size and the number of cut sites.
Fragments, approximately less than about 50-100 kb, e.g., 90 kb,
frequently relax completely to form balls as shown in Figs. 13B,
C and F and 15. Restriction digestion results that vary in
fragment number, size and order can be used to readily
characterize a mixture of similarly sized but different
molecules. Figure 13C-D, and Fig. 13E and F, show results
obtained from two such chromosomal mixtures. In the first
example, a small distal restriction fragment on chromosome 5
(Fig. 13C) serves to differentiate it from chromosome 8. The
second example (Fig. 13E and F) shows that a single cut can
differentiate similarly sized molecules, given sufficient
resolution. These shear-sensitive, megabase sized DNA molecules
can be mounted with minor breakage and mapped by means of a
lower power (e.g., 40-80X,e.g., 63X, as compared with 80-500X)
microscope objective.
Figures 14 and 15 show an ordered restriction map (and
corresponding fluorescence micrographs) created from a Csp I
digestion of chromosome 9 by optical mapping as compared to maps
created by pulsed-field gel electrophoresis (PFGE) and
hybridization with a series of genetically mapped sequences.
Chromosomes 3 and 11 are as known in the art. To avoid possible
prejudical selection and procesing of image data, optical maps
can be made and then checked with electrophoretically derived
data. The overall agreement between the optical and pulsed
electrophoresis maps is expected to be and found to be generally
excellent.
Large-scale maps are rarely made without any errors.
Maps created by optical mapping may contain some errors that
stem from incorrect fragment number determination or from the
stated limits on precision of our fragment-sizing methods. DNAs
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that have nearly symmetric maps cannot be optimally averaged to
improve resolution unless one end is identified, so that map
polarity must be established through ancillary means. Given
expected levels of sizing precision, fragments can be detected
above 10, 20, 31, 40, 50, 60, 70, 80, 90, 100, 151 or any range
or value therein.
Optical mapping of this invention can be extended to
mammalian genomes by technical advances that permit the
detection and quantitation of restriction fragments (300-
100,000, or 500 to 10,000 bp or any range or value therein)
generated by frequent-cutting enzymes. Ordered contigs of well-
characterized fragments can then be constructed from high-
resolution, ordered restriction maps created from randomly
sheared genomic DNA, e.g., 200-2000 kb in size or any range or
value therein. YACs or cosmids can also be similarly analyzed
and compared with the genomic map, to facilitate ordering.
Engineering changes in chamber design, sample handling, image
analysis, and informatics provide a high throughput methodology
that is capable of rapidly mapping entire genomes and, more
importantly, extending knowledge of sequence information to
populations of individuals rather than to a prototype of each
organism.
An eighth preferred embodiment of this invention
relates to mapping nucliec acid sequences using hybridized
probes. With this technique, a nucleic acid, a probe (a
characterized nucleic acid) and under certain circumstances, a
recombinational enzyme, are combined in a matrix. The probe may
be of any practical length and may be labelled with any suitable
labelling agent. If a restriction enzyme is used, (e.g., if the
probe is not capable of invading the target molecule without the
use of a restriction enzyme) it may be any suitable enzyme, for
example, one known in the art for conventional labelling of DNA
probes. As a non-limiting, specific example, a useful
restriction enzyme is recA.
Hybridization can be initiated by any suitable means,
for example, by diffusing ATP and magnesium ions into the
microscope slide.
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Probes can be hybridized to a target molecule and
visualized in at least two different ways. First, the probe may
be visualized directly if it is sufficiently large. For
example, a probe larger than 1 kb probably can be visualized
using microscopy equipment which is currently available.
Second, a chromophore, or other suitable labelling agent can be
attached to the probe and can be detected visually or
spectroscopically. For example, Texas Red or rhodamine, as well
as other chromophores, may be used.
After the probe has been hybridized to the target
molecule, there are at least two preferred ways to map the
position of the probe. However, other methods are also within
the scope of this invention. As a first preferred alternative,
the curvilinear length of the target molecule can be measured.
When a light microscope is used, fluorescence intensity measure-
ments can be used to locate and quantify linear regions which
are not totally stretched out. The position of the probe is
located based upon its characterizing feature, e.g.,
chromophore, radioactive tag, and/or size. As a second
preferred alternative, a target molecule is cut with restriction
enzymes and all of the fragments are sized and measured by the
methods of this invention. The location of the hybridized
fragment is determined by one of the methods described above,
e.g., by direct visualization of the molecule or by microscopy
combined with spectroscopic technique. All of the fragments are
sized, and the distance from the labelled fragment to either end
of the target molecule is then easily calculated. The exact
position of the probe on a particular restriction digest can be
mapped with the addition of a new enzyme for further digestion,
and these fragments can then be mapped visually.
A preferred approach to optical mapping of the present
invention is imaging stained, single, deproteinized DNA
molecules during restriction enzyme digestion allowing direct,
ordered mapping of restriction sites. In brief, a flow field
(or other type of field) is used to elongate DNA molecules
dissolved in molten agarose and fix them in place during
gelation. The gelation process restrains elongated molecules
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~s
from appreciable relaxing to a random coil conformation during
enzymatic cleavage. A restriction enzyme is added to the molten
agarose-DNA mixture and cutting is triggered by magnesium ions
diffused into the gelled mixture (mounted on a microscope
slide). Cleavage sites can be visualized as growing gaps in
imaged molecules. The resulting fragments are sized in two
ways: by measuring the relative fluorescence intensities of the
produces, and by measuring the relative apparent DNA molecular
lengths in the fixating gel. Maps can be subsequently,
constructed by recording the order of the sized fragment,
averaging a small number of molecules rather than utilizing only
one improves accuracy and permits rejection of unwanted
molecules. Optical map production is very rapid because of the
combination of restriction fragment ordering in real time with
fast accurate sizing techniques. Optical mapping this provides
a powerful new technology for rapidly creating ordered
restriction maps of lower or higher eucaryotic chromosomes or
YACs, without the need for analytical electrophoresis, cloned
libraries, probes, or PCR primers. Incremental technical
improvements should enable the rapid high resolution mapping of
mammalian chromosomes and ordering of YACs.
In optical mapping, in which stained, single,
deproteinized DNA molecules are imaged during restriction enzyme
digestion to allow direct, ordered mapping of restriction sites.
To briefly describe the steps involved in optical mapping, a
flow field (or, in principle, an electrical or other field) is
used to stretch out DNA molecules dissolved in molten agarose
and fix them in place during gelation. The gelation process
restrains elongated molecules from appreciably relaxing to a
random coil conformation during enzymatic cleavage. The
activity of a restriction enzyme already present in the agarose-
DNA mixture is triggered by magnesium ions diffused into the
gelled mixture (mounted on a microscope slide). Cleavage sites
are visualized as growing gaps in imaged molecules. The
resulting fragments are sized in two ways: by z,'aasuring the
relative fluorescence intensities of the products and by
measuring the relative apparent DNA molecular lengths in the
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fixating gel. Maps are then constructed by simply recording the
order of the sized fragments (see Section 3.2.vii). Averaging a
small number of molecules rather than utilizing only one
improves accuracy and permits rejection of unwanted molecules or
fragments. Optical map production is very rapid because of the
combination of restriction fragment ordering in real time with
fast accurate sizing techniques. This approach opens up a
powerful new technology for rapidly creating ordered restriction
maps of lower eucaryotic chromosomes or YACs, without the need
for analytical electrophoresis, cloned libraries, probes, or PCR
primers.
Optimization of optical mapping for genomic analysis
includes alternative molecular fixation techniques; new
molecular sizing techniques that do not rely on analytical
electrophoresis; new image processing approaches, tailored to
mapping needs, and 4) new approaches for restriction map
construction, which alternatives are now provided by the present
invention. These efforts are described in the following
subsections.
(i) Gel fixation and mechanics of DNA relaxation under
tension and cleavage: A single large DNA molecule 200 nm long
(600 kb) is a random coil in solution which can be visualized as
a loosely packed ball averaging 8 nm across (61). Optical
mapping begins with stretching out such a DNA molecule and
fixing it in place to inhibit rapid relaxation, prior to imaging
by light microscopy. The fixed molecule must lie within a
shallow plane of focus for successful imaging. Elongated
molecules in a gel behave mechanically like a stretched spring:
fixed molecules are under tension which is released during coil
relaxation to a random conformation. DNA molecules embedded in
agarose gel, have been modeled during electrophoresis, as a
series of connected pools of coil segments under tension with
each other, and calculates that the force (f;) associated with
the free energy change of shuttling coil segments between pools
is given by:
f; = 3kT/ (2n;b) ( (aZ/n;b2) -1) + (kT/b) lnC
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where k is the Boltzmann constant, a is the gel pore diameter, n;
is the number of associated coil segments, b is the coil segment
length, T is the temperature and C is a constant relating to
coil segment structure. This result shows that the tension
developed between pools is inversely related to the number of
segments contained within a pore volume. It follows that a
stretched-out, elongated molecule is under more tension than a
compact, relaxed one.
Large DNA molecules are conveniently stretched out in
molten agarose by flow forces and then rapidly fixed in place by
agarose gelation, without applying electrical fields.
Experimentally, the kinetics of gelation are controlled by
temperature, and optimization of the annealing conditions. for
our analysis, DNA coils must be critically stretched: too much
and the molecule becomes difficult to image; too little, and
there is insufficient tension to reveal cut sites. Excessively
stretched molecules present too little fluorochrome per imaging
pixel, so that measured molecular intensities approach
background values. Additionally, the fixation process has to be
gentle enough to permit some coil slippage to reveal cut sites.
Taking these and other considerations into account, one can
practically optimize fixation conditions to produce molecules
spanning approximately 20% of their curvilinear contour lengths.
Restriction Digestion of Single Molecules Including
Optical Mapping detects restriction enzyme cleavage sites as
gaps that appear in a fixed molecule while fragments relax to a
more random conformation. Since the rates of enzymatic cleavage
by different restriction enzymes are variable (64), careful
adjustment of the timing is critical. Cleavage should occur
only after molecular fixation is complete because premature
reactions disrupt attempts to phase fragments. This timing
problem can be solved by premixing the agarose-DNA solution with
restriction enzyme, at 37 C, and triggering the reaction by
diffusing magnesium ions into the viewing field, without
disturbing the gel. Aside from gaps, cleavage is also signaled
by the appearance of bright condensed pools or "balls" of DNA on
the fragment ends at the cut site. These balls form shortly
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after cleavage and result from coil relaxation which is favored
at ends. This pooling of segments is useful in map making
because it helps to differentiate out-of-focus segments, that
might appear as gaps, from actual cuts. Cleavage is scored
reliably by both the appearance of growing gaps and enlarging
bright pools of segments at the cut site.
Map Construction - Fragment Number Determination: As
described herein, large scale restriction maps have been
constructed primarily from electrophoretically derived data. In
contrast, the present invention involves as the first step
determining the number of cleavage sites within a molecule. The
cut sites within a molecule tend to appear at irregular times
after Mg2+ addition. All possible cleavage sites may not appear
simultaneously; instead, cuts usually appear within 5 minutes of
each other, e.g., under the conditions described herein. The
extent of digestion depends on a number of factors including
both the fragment number and size. The correct number of
cleavage sites may be determined by histogram analysis of
partial digestion results (molecules are sorted, binned and
counted by the number of cuts). Typically, 10-20 molecules
suffice for this analysis, and the bin containing the largest
number of cut sites whose molecules can be properly averaged by
intensity and length measurements for size, can provide the
correct number.
Fragment Sizing By Relative Intensity: The second
step can be to size the resulting restriction fragments. For
this purpose two complementary approaches can be used, one based
on relative fragment fluorescence intensity and the second on
apparent relative length measurements. However, neither
approach may provide absolute values, but each can be
standardized readily. The gel fixation technique described
above also produces a natural substrate for intensity
measurements since an entire molecule can be brought into focus.
Gel fixation is able to flatten molecules spanning as much as
250 microns. Segments of molecules that are out of focus cannot
be used for intensity measurements because theiY intensities are
not proportional to mass in any simple way. A relevant
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observation here is that when an elongated molecule
substantially relaxes, most of its mass moves out of focus, as
expected, since the hydrodynamic diameter of a fully relaxed 700
kb DNA molecule in fluid is 8 microns while the depth of focus
used for imaging molecules under the microscope is approximately
0.2 micron.
The absolute fluorescence intensity of a DNA fragment
in the microscope is determined by any one or more of many
variables, such as the camera gain control and lamp brightness,
and therefore is not a desirable quantity to measure. By
calculating the relative intensity of two fragments (from the
same parental molecule), the fragments are allowed to serve as
an internal intensity reference for the other. Relative
intensities are converted to kb by multiplying by the known or
independently determined chromosome size.
Other Methodologies for Sizing, Manipulating and
Characterizing Single DNA Molecules
Mapping Known Sequences using optical mapping.
According to the present invention, specific sequences can be
localized on single DNA previously proposed using RecA protein
to D-loop chromophore-labeled homologous sequences into large
target DNA molecules, followed by optical mapping, to accurately
position this complex. However, with the development of the
Achilles heel (66) technique and especially RARE (67, 68), a
RATE technique which uses the RecA-probe-target complex to block
EcoRI methylase action can also be used in methods of the
present invention. Removal of the complex leaves a target
molecule vulnerable to EcoRI cleavage at only the unmethylated
site(s). Obviously, the RARE technique can cleave DNA at any
location, given known sequences. Developed a type of high
resolution FISH, since the target is a stretched-out naked DNA
molecules instead of chromatin.
Optical mapping can be combined with RARE to create
high resolution maps of lower eucaryotic chromosomes, or YACs.
Fig. 8 shows results of cleaving yeast chromosomal DNA
molecules. Oligonucleotides containing 50 bp from the yeast
LEU2 and ERG16 genes were synthesized and used for RARE analysis
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on the microscope. Shown in Fig. 8A is a RARE mediated cleavage
of Candida albicans Chromosome 5 DNA using an ERG16
oligonucleotide (69). Comparing the optical mapping data
obtained from intensity ration in RARE mediated cleavage of S.
cerevisiae Chromosome III, where fragments of 110 and 235 kb
were obtained (the fluorescent intensity ratios of five
molecules were binned and averaged), electrophoretically derived
data yield 100 and 245 kb fragments. The published map for
Candida albicans that is electrophoretically derived produced
180 and 1020 kb sized fragments compared to the optical mapping
data of 250 and 950 kb; six molecules were averaged for this
optical mapping.
RARE, combined with optical mapping, according to the
present invention provides a very powerful combination for rapid
restriction mapping of the human genome. RARE can be used to
first excise specific mammalian genomic regions, which are
puried using pulsed electrophoresis. Then, e.g., optical
mapping can be used to create a restriction map for the defined
genomic region.
Using Single Molecule for Sizing.
Sizing Molecules by Coil Relaxation Dynamics.
Fluorescence microscopy has been used to study the
conformational dynamics of single DNA molecules during gel
electrophoresis (53, 59, 70). Studies, both experimental and
theoretical, of DNA conformation during gel electrophoresis show
that a DNA molecule stretches out to form long hooks, which
relax back to a compact conformation, in a cyclically occurring
fashion (19, 53, 63, 71). Hook formation can be used to stretch
DNA molecules out so that when the perturbing electrical field
is shut off, relaxation kinetics of single molecules can be
quantified by simply imaging them and measuring length changes.
This measurement is similar to stretching out a spring,
releasing it and monitoring the recoil kinetics by watching it
shrink back to a relaxed coiled state. Traditionally, the
viscoelastic technique (61) has been used to measure relaxation
time (7) so that molecular weights could be determined.
However, a severe limitation is imposed on such molecular weight
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determinations, only the largest molecules in solution can be
sized; a spectrum of sizes cannot be measured.
Parallel measurements can be made using molecular
imaging techniques (e.g., fluorescence microscopy), and size
distributions can be determined since the conformational
dynamics of each molecule is measured separately. There is
another compelling reason for studying relaxation kinetics: the
associated relaxation time (T) are strongly size dependent, with
T proportional to (molecular weight) 1=5'3, so that size
discrimination is excellent. It surpasses any other sizing
technique with the exception of sequencing. The determined size
dependence will vary w---h the chosen relaxation mode.
The fast coil relaxation times that correspond to
Zimm-Rouse relations (72, 73) normally encountered in solution
can be initially measured. In a gel matrix, a stretched out DNA
molecule with length L(t) (this is actually the length of the
primitive tube) will relax as <L(t)>=Aexp(-t/r)+<Le>(74, 75),
where T is the relations time, t is time and the brackets
represent an ensemble average. L(t) is not the molecular
contour length, but it can be interpreted here as the apparent
molecular length as imaged by the microscope. Le is the
equilibrium molecular tube length and is measured as a plateau
region in an exponential decay. L forms the basis of the
"Baseline" sizing methodology, as discussed herein.
Measurements of fast coil relaxation times are simple
to carry out. Large DNA molecules, stained with ethidiuin
bromide, are embedded in 1t agarose and mounted on a
epifluorescence microscope, equipped with a SIT camera (a low
light level sensitive device) and interfaced to an imaging board
set contained within a computer. Electrodes in the microscope
chamber are pulsed so that molecules form hooks, and their
lengths are measured automatically during relaxation by a
special program written in NIH image (Wayne Rasband)
macroprogramming language. The relaxation of the DNA molecules
starts when the applied field is shut off. Single exponential
relaxation times are calculated for a series of yeast
chromosomal DNAs and are graphed as shown in Fig. 9 (47), as a
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In-In plot versus size (a Mark-Houwink plot). The slope of this
line gives the molecular weight dependency for T, the relaxation
time (T) =constant (size)1-45 (kb)
Sizing DNA Molecules by Baseline Measurement.
Typical DNA relaxation plots, as apparent length versus time,
provide plotted points which are averages of 4 relaxations.
Such a plot shows that the measured length decreases in an
exponential fashion and importantly that the molecule does not
fully relax to a spherical random conformation. Instead, the
quasiequilibrium structure is a thickened, short rod-like
object, which signals an end of the exponential decay, and its
length is the baseline for the plot. Additionally, very slow
relaxation processes are still happening, but they are of a
different nature and time scale, which could be proportional to
mass3. Within the time scale used (e.g., hundreds of seconds),
length measurements settle down to an equilibrium value which is
termed the "baseline". Baseline values vary linearly with DNA
size and are very reproducible. Thus, a relaxation measurement
yields sizes in two ways: 1) by determination of the relaxation
time, T, and 2) by baseline determination.
Optical Contour Maximization. DNA molecules can be
almost totally elongated by the action of a relatively weak
electrical field if one end of the DNA is fixed (59, 76, 77).
Length is simply measured by imaging the elongated molecule
using fluorescence microscopy. However, fixing large linear DNA
molecules is cumbersome, and previous elongation studies did not
attempt to correlate length with molecular mass in any
systematic manner.
A practical sizing methodology, termed Optical Contour
Maximization (OCM), is based on the discovery that when a linear
DNA molecules snags an obstacle during electrophoresis in a
loose gel matrix, it elongates nearly completely to form a
metastable hook that can persist for several seconds (46). This
loose matrix can be found at the coverslip-agarose gel
interface. The longest observed hook contour length can be
determined from rapidly collected images and that these maximal
lengths which show a linear correlation with respect to reported
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size (240-680 kb; (46)). To summarize, OCM provides very
precise size measurements that are superior to most results
derived from analysis by pulsed field electrophoresis.
Spectroscopic Studies of DNA Molecules in a Gel:
A fluorescence dichroism spectrometer (similar to (78-
80)) can be constructed that is attached to an inverted
microscope; the microscope can be also equipped with a SIT video
camera and interfaced to a video-digitizing board housed in a
computer. A major advantage of such spectroscopic
instrumentation, in contrast to that of Boredjo and Holtzwarth
(78-80), is that molecules can be imaged, as well as collecting
spectroscopic data on a small number of molecules using the
microscope-spectrometer combination. Imaging and spectroscopic
data are somewhat complementary, and combining them allows
almost completely characterization of an electrophoretic system.
Consider that fluorescence dichroism monitors the average coil
orientation, at a resolution that is beyond the Rayleigh limit
(approximately 1 gel pore in size), whereas, imaging experiments
elucidate gross coil conformation dynamics. Such an instrument
can be used in this invention to correlate the imaged molecular
dynamics of large DNA molecules during electrophoresis with
their change in dichroic ratio.
As described herein, optical mapping is a new physical
mapping approach that encompasses many new single macromolecular
phenomena and which results in rapid construction of ordered
restriction maps from single DNAs. Restriction fragments are
sized optically with minimal perturbation, and multiple
molecules can be analyzed in parallel.
The following methods used in the present invention
provide improved sizing capabilities. New methods aimed at
reducing or eliminating apparent partial digestion are also
provided.
Methods for Fixing Single DNA Molecules for Light
Microscopy
optical mapping analyzes large naked DNA molecules,
typically microns in diameter, that exist in solution as random
coils. These molecules must be stretched out and fixed in place
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before image separation can be performed of fragments resulting
from site-specific cleavage. Molten agarose is used to supply a
fluid flow to stretch large DNA molecules and subsequent
gelation to fix them in place. Gel fixation is firm enough to
hold molecules in place, but gentle enough to permit some
slippage upon cleavage so that cut sites are observable.
Indeed, how agarose is able to fix large DNA molecules is not
well understood. A more reproducible fixation can also be
obtained with regards to controlling the level of elongation, to
preventing premature relaxation and to allow more reliable
detection of cleavage sites when formed, and to improve
throughput.
(i) Understanding and characterizing the gel fixation
process: Gel fixation is a fundamental component of optical
mapping, yet how agarose traps and fixes molecules is poorly
understood. It is not even certain that agarose fibers are
physically locking and entrapping DNA molecules, thereby fixing
them in place. Gel fiber-DNA interactions may also play a
prominent role in DNA gel electrophoresis, as discussed herein.
Indeed, full characterization of the mechanisms of gel fixation
will lead to improved molecular fixation techniques that should
be applicable to a broad range of single molecule methodologies
beyond optical mapping. As described herein, gel fixation of
elongated molecules is expected to result from a combination of
gel fiber-DNA interaction and relaxation effects, particularly
at the ends of molecules.
Such different interactions can be characterized by
studying the DNA segment distribution in a series of samples of
gel-fixed molecules that vary in the size of the DNAs and in the
gel concentration. Intensity measurement techniques developed
for optical mapping of the present invention can also be used to
create intensity profiles of fixed, single DNA molecules along
the major axis as a function of time after mounting (see Fig.
10), also of the present invention. By analyzing these
intensity profiles as they vary with time, trapping points can
be located and/or characterized along the embedded molecule as
regions of intensity invariance. In other words, as a fixed,
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elongated coil attempts to relax, coil segments move in and out
of pore volumes. If there is an obstruction, i.e., strong
fiber-DNA interaction, coil segments will not be able to relax
past that position. To further evaluate the degree of trapping,
the system can be perturbed with a carefully measured electrical
field and image resulting motions, with accompanying intensity
measurements. From these analyses, the mean number of trapping
points per kb of DNA can be determined as a function of the
total coil size and gel concentration. The effects of ionic
strength and divalent counter ions on fixation can also be
measured.
Gel Concentration Effects: The gel concentration used
for optical mapping has not been optimized; but 0.1-3t LGT (low
gelling temperature) agarose, or any range or value therein can
be used, such as lg LGT agarose. Since gelation fixes embedded
molecules, systematically varying the gel concentration should
modulate the degree of fixation and ultimately the rate of
molecule relaxation. For example, a higher gel concentration
may be desired for small fragments as compared with larger ones,
this would serve to retard relaxation and facilitate our
analyses of these smaller,fragments. Additionally, gel
concentration affects gelation kinetics so that smaller
molecules are trapped in an extended conformation before
substantial relaxation takes place. Such kinetics and their
effects on trapping can be determined by systematically vary gel
concentration for a range of DNA sizes (30-1,000 kb) and
studying its effect on stabilizing molecules against premature
relaxation and on apparent cutting efficiency. Molecular
relaxation are evaluated by measuring lengths as function of
time, as descibed herein, and cuts are scored using optical
mapping procedures.
(iii) Adhering Large DNA Molecules to Glass: Yanagida
showed that short elongated DNA molecules could be adhered to
glass in the presence of Mg+Z and imaged by fluorescence
microscopy (81). The adhesion was stable enough to permit film
exposures, lasting several minutes, of individual fluorochrome
stained DNA molecules. This technique can be adapted according
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to the present invention in order to spread and fix large DNA
molecules to magnesium ion "loaded" glass slides (loaded with
Mg+z by soaking glass slides in magnesium chloride solution).
Our procedure consists of the following: spermine condensed
molecules obtained from agarase treated electrophoresis gel
slices of separated yeast chromosomes, where the agarase has
digested agarose into small saccharides, is spread in a thin
layer over the magnesium loaded glass. Spermine condensation
can be included to substantially prevent shear-induced breakage.
The spermine concentration is controlled so that the available
magnesium in the loaded glass can decondense the molecule and at
the same time adhere it to the glass surface. Subsequent
optical mapping is carried out with the modification that
restriction enzyme is added to the glass-fixed molecule, instead
of Mg+2 being added as conventionally occurs. If cut sites do
not appear readily due to firm fixation conditions, then adding
a small amount of EDTA helps loosen overly fixed molecules.
Glass fixed molecules possess several advantages over
their gel-fixed counterparts: 1) since molecules are attached to
the cover slip, images are very sharp and bright (no gel to
scatter and obscure); 2) images have less fluorescence
background in the field, agarose gel is a relatively crude
product with extraneous fluorescence; 3) bright, clear images
are easy to process by fully automated routines, and 4) fixation
can be tighter so that molecules can be more effectively spread
out. This last advantage is a critical requirement for
producing higher resolution maps, which naturally consist of
more numerous, smaller fragments.
(iv) Using Electrical Fields to Spread and Fix
Molecules: DNA molecules can stretch out and elongate
considerably during gel electrophoresis, as has been shown by
fluorescence microscopy (53, 62). Although electrical fields
elongate molecules during electrophoresis, it not obvious how to
maintain this state in the absence of any applied field. In
fact, trying to maintain elongation during gel electrophoresis
is antithetical to separation mechanisms which require some
relaxation processes to size resolve DNA molecules.
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From our trapping studies, e.g., as described herein,
segments of electrophoresing coils have been imaged that serve
to trap or anchor a whole molecule in place. High electrical
field strength can be used to form these anchors during agarose
gel electrophoresis, and these anchors can persist long after
the field is removed, e.g., for an hour or more. Trapped, fixed
molecules are useless for electrophoresis, but are just the
molecular conformation suitable for optical mapping.
Exploitation of such conditions that trap molecules
unproductively for electrophoresis, productively trap molecules
for our optical mapping of the present invention. Accordingly,
systematically varying of field strength conditions for a range
of molecular sizes (200-1000 kb), as well as measuring the
resulting lengths and elongated state persistence times, can be
used according to the present invention for analytical
techniques of optical mapping, as described herein. Agarose gel
concentration can also be systematically varied. Thus, optimal
elongation conditions for optical mapping can be established.
Single Molecule Sizing Methodologies. Optical mapping
is dependent on methodologies for sizing single molecules and
construction of restriction maps of higher resolution and
precision can be provided by such methods of the present
invention. As non-limiting examples, five single molecule
sizing methods are provided, including apparent relative
length, fluorescence intensity ratio, relaxation in a gel,
baseline, and OCM (optical contour mapping). These can be
usefully classified into two groups: techniques that require
molecular perturbation and those that require none. Non-
perturbation sizing techniques such as measurements of
fluorescence intensity of single molecules and apparent length
measurements are convenient to use because they do not require a
sophisticated microscope mounted chamber and attending
controlling electronics. Additionally, non-perturbing sizing
techniques are also well suited for parallel measurements.
Despite some of these advantages for the non-perturbing class,
the perturbation-based sizing techniques of the present
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invention provide unexpectedly superior results, since they
provide high precision and resolution.
Several approaches for optically sizing DNA fragments
of at least 500 bp can be provided by the present invention,
e.g., as follows.
Apparent Relative Length and Intensity Measurements:
As described herein, ordered chromosome restriction maps can be
generated according to the present invention by using relative
intensity and relative length measurements. Molecules are first
elongated and fixed in agarose gel containing restriction
enzyme. Magnesium ions are then diffused in, triggering
digestion, and restriction sites are visualized as growing gaps
in the DNA molecules. This approach is simple, effective, and
has superior sensitivity, since one molecule can be visualized
directly. The microscope chamber may consists of a slide with a
hole drilled in it, gel and a cover slip. Although no
electrodes are used to apply electrical fields to stretch or
manipulate the molecules, it is possible that applying
electrical fields could beneficially perturb the system.
Consider that both relative apparent length and relative
intensity sizing methodologies require that fragments remain
elongated for optimum results. Gel fixation is not perfect, and
fragments suboptimally fixed are prone to premature relaxation
which can complicate sizing attempts. Furthermore, fixation of
the DNA to the gel matrix can interfere with the observation of
cut sites, which requires local relaxation to produce visible
gaps.
Bearing the above considerations in mind, electrical
fields can be used to perturb molecules during optical mapping
so that more usable and more precise data can result from a
single mount. An electrical field can be controlled to stretch
or move DNA molecules; these perturbations will elongated
relaxed molecules and allow relative intensity and length
measurements. Perturbations should also reveal apparently
partially cut molecules so that cut sites, present but not
otherwise visualized as gaps, appear. Recent preliminary data
from our laboratory show that, indeed, electrical fields do
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,,.~~:
decrease the degree of apparent partial digestion. Electrical
perturbations are implemented during optical mapping by using
the chamber. The only additional modification is to totally
fill the chamber with agarose and DNA.
Alternate Approaches to Contour Lengths Measurements:
a sizing methodology, termed Optical Contour Maximization (OCM
(46)), which transiently stretches linear DNA molecules out in a
loose gel matrix using an applied electrical field and then
sizes them by optical length measurement. Molecules stretch out
by snaring on obstacles in the loose matrix to form hook-like
conformations. Remarkably, a relatively weak electrical field
(e.g., 5-30, e.g., 20 volts/cm) is sufficient for complete
elongation of a tethered or temporarily snared DNA molecule (46,
59, 77). If the hook arms are similarly sized, then the
molecule can be stretched out to nearly its full contour length.
OCM sizing accuracy and precision is very high, as good or
better than pulsed electrophoresis based measurements.
OCM can be modified as known to provide high
throughput methodology of the present invention by using a new
physical effect to elongate molecules and new iinage processing
methods to measure molecular lengths in real time. A fluid-gel
interface, which is easily made, provides an ideal situation for
differential frictional forces to act on an electrophoresing
molecule and elongate it to nearly its full contour length, just
like in OCM. The net elongation force on the molecule is
determined by the differences in DNA frictional coefficient in
the gel matrix versus the fluid phase. More precisely, when a
DNA molecule electrophoreses through a gel-fluid interface, as
depicted in Fig. 11, the fluid frictional forces are much less
than those posed in the gel matrix. These forces are,
typically, at least 10-fold less (55), and differences vary with
gel concentration. Molecular conformation is dynamic within the
gel matrix, but on the average it is relatively compact.
Frictional forces are reduced when a molecule merges from the
gel matrix into the free solution presenting a differential
force across the molecules sufficient enough to cause it to
elongate. Immediately after a molecule completely pulls free of
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the matrix, elongation forces disappear, and the molecule
relaxes back to a compact, free solution conformation.
Reversing the electrical field sends the free molecule back into
the gel matrix; the length measurements can then be repeated and
averaged as many times as needed, depending upon the desired
accuracy.
High throughput is accomplished by electrophoresing
molecules through the interface at a rate of approximately 20-50
molecules/minute. Contour lengths can be measured and tabulated
from stored data by the same techniques and computer algorithms
developed for optical mapping and coil relaxation measurements,
imaged, such as the non-limiting example of images from a SIT
camera are rapidly digitized, frame averaged and stored as 16
bit files at a frame rate of 30/sec. For example, 120 file
frame buffers can be used in the analyzing computer. This means
that 120, 512x512 pixel images can be digitized and stored in as
little time as 4 seconds. More rapid image storage is available
by simply reducing image size, in which case the same hardware
can store 480, 128x128 pixel images. Processing algorithms can
thus size 5-10 molecules simultaneously by gathering
approximately 10 images (averaging 4-16 frames together) in a 20
second interval. Using a 1 gigabyte hard disk for storage means
close to 2,000 full frame images or sizing data for 1,000-2,000
molecules can be stored. Processing algorithms can be set up to
work in batch mode and require approximately 3-5 hours to
process 1 gigabyte worth of image data into 1,000-2,000 sizes
tabulated on a spreadsheet. These processing times are based on
unattended operation, but operator interfaces can also be used
that permit convenient manual identification and marking of
molecules for analysis.
High image quality greatly facilitates image
processing. Fluorescence images of DNAs obtained in fluid
rather than gel are brighter, sharper and relatively free of
fluorescing artifacts. Consequently, they are ideal for
unattended image processing since they can be transformed into
reliable binary or digital images, which are easily accepted by
our processing algorithms. This high throughput sizing
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methodology can be tested and benchmarked by using a series of
Not I digested yeast chromosomes mixtures (containing DNAs 30-
900 kb), of increasing complexity. Statistical analysis to
calculate the precision of single measurements can be performed
and the ultimate accuracy of this methodology determined.
Confidence intervals are determined to establish the minimum
number of molecules necessary for adequate analysis of complex
mixtures. This analysis will help determine the usable size
resolution and size discrimination levels. Sources of noise and
systematic error are detected and eliminated as much as
possible. A lower size limit of 5-20 kb and an increased upper
size limit are provided by the present invention since molecules
with contour lengths greater than the microscope viewing field
are sized by offsetting a known distance from the interface and
monitoring only coil ends.
A Fluid Interface System for Sizing by Coil
Relaxation: As discussed herein, coil relaxation is quantitated
in gel and derived mathematical relationships for molecular size
based on our theoretical and experimental calculations and
observations. This measured relaxation time is very sensitive
to molecular weight and varies as (molecular weight)'-', whereas
length measurements vary only as (molecular weight)1 at their
best. Within a given size distribution the largest molecules
dominate the measured relaxation, so that the smaller molecules
in heterogeneous mixtures cannot be analyzed in a chamber (83).
Unfortunately, the inability of this method to determine a size
distribution made it impractical for use in most standard
analyses in molecular biology. Under the microscope, of course,
individual chains are observed independently, effectively
deconvoluting any mixture.
Coil relaxation can be measured according to the
present invention in free solution using the described gel-fluid
interface system in free solution for providing rapid and
sensitive techniques for size determination of heterogeneous
samples, a continuation of our present work. Yanagida and
colleagues were able to measure coil conformational dynamics of
single molecules in solution, using fluorescence microscopy,
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yielding reliable average molecular dimensions that can easily
be related to size (44). Other optical methods such as electric
birefringence (84) and dichroism (85) have also determined the
same weight dependence on relaxation as the shear based,
viscoelastic technique.
The gel-fluid interface system offers an almost ideal
set of conditions for relaxation measurements in free solution:
1) the degree of elongation can be easily controlled by varying
the electrical field strength or the gel concentration; 2)
elongation conditions are uniform and reproducible; 3) repeated
measurements are simple since reversing the field permits re-
extraction of molecules from the interface, and 4) overall,
experimental conditions are controllable and measurements take
place in a well-defined field of view, an important
consideration in microscopy.
Relaxation times are determined by electrophoresing
molecules through the gel-fluid interface described in the
previous section (see Fig. 11A-C) and initially using the same
techniques developed for gel-based relaxation measurements.
These determinations use optical length measurements at periodic
intervals to quantitate the degree of relaxation. The change of
measured molecular lengths as a function of time are fitted to a
single exponential decay to obtain the relaxation time.
Solution relaxation mechanisms are somewhat different than gel-
based ones, in that coil segments are not confined to move
within a tube, or a series of connected gel pores. In free
solution, elongated DNA molecules relax by evolving from a
drawn-out prolate ellipsoid to a more symmetric, spherical
conformation (44). Relaxation times are also shorter in free
solution (generally 10-fold less, (55)): for example, a 500 kb
molecule has a relaxation time of 4 seconds. However, since
solution relaxation times are inversely proportional to solution
viscosity, measurements on small molecules can be made on a
convenient time scale by simply adding glycerol or sucrose to
increase viscosity. it is significant that the shorter
relaxation times manifested by solution based relaxation
measurements are advantageous for any high throughput approach.
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Image collection procedures are virtually identical to
those described in the previous section and the same images can
be used for length and relaxation measurements. Image
processing routine can be modified to fit ellipsoids around the
relaxing coil mass, and the associated major and minor axes are
used to measure relaxation progress. As described in the
previous section, a set of molecules are used to benchmark and
establish relaxation dependent sizing conditions. Statistical
analysis can be used to determine the precision and accuracy of
these measurements.
Providing the Gel-Fluid Interface System for
Fluorescence Intensity Measurements: A single molecule
fluorescence intensity method can be used to measure the
relative fluorescence intensities of restriction fragments
embedded in agarose gel that form during optical mapping. The
relative fluorescence intensity of a restriction fragment
correlates well with its relative mass and have used these
measurements to construct preliminary Optical Maps for several
chromosomes of Saccharomyces cerevisiae. In general,
fluorescence intensity measurements are difficult to quantitate
on a microscope-derived image, so that relative measurements
provide a sort of internal standard. There are several
drawbacks to this approach since errors are absolute instead of
being relative. This means that, for example, a 20 kb standard
deviation applies equally to a 60 kb fragment as well as to a
900 kb sized one. In other words, the coefficient of variation
(mean/standard deviation) can vary enormously and will penalize
small fragments more than larger ones.
The gel-fluid interface system can be used in this
invention to obtain high resolution fluorescence intensity
measurements of single molecules. Measurement precision can be
increased and lower size limitations made for these
measurements. Increased sizing performance accrues from a
number of factors, such as, but not limited to, the simultaneous
use of three sizing methods will increase precision and accuracy
(length, relaxation and intensity); the fluid-gel interface
system produces optically flat molecules more consistently than
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gel fixation; the degree of molecular elongation is more
controllable than gel fixation, and the elimination of gel
provides sharper, brighter images that are more free of
fluorescing artifacts. As mentioned previously, high quality
images are easier to image process automatically.
Intensity quantitation requires that molecules be in
focus. The gel-fluid interface system creates elongated
molecules which are optically flat. More precisely, a high
power, high numeric cal aperture objective has a depth of focus
approximately 0.2 microns deep (86), so that for a molecule to
be optically flat, or totally in focus, it must lie completely
within a box with a thickness comparable to this value.
Since molecules are continuously electrophoresed in
the gel-fluid interface system, intensity measurements must be
made on moving targets. A potential problem is that images must
be grabbed quickly to avoid serious motion-induced distortion,
and time is limited for frame averaging. However, for short
observation times, higher illumination levels (providing greater
signal to noise) can be used without significant breakage or
bleaching. Frame averaging can be used to reduce noise and
increase the number of gray levels. For example, we use a 8 bit
digitizer with a 16 bit frame buffer and average 32 frames,
which is approximately 1 second of video. This gives 13 bit
images containing almost 8,200 levels of intensity. If images
are grabbed more quickly, the resultant images contain fewer
levels of intensity. Shutting off the electrical field is one
way to increase image averaging time. However, molecules begin
to relax immediately and move partially out of focus.
Motion problems can be solved by initiating imaging
while the molecule is still partially embedded in the agarose.
At this point, the molecule is optically flattened and is not
moving rapidly since it is still embedded in the agarose matrix.
To ensure that long molecules are completely in view, the
electrical field can be varied to produce different degrees of
elongation. Using computer-interfaced programmable power
supplies, ramping the field strength systematically can also be
tested to provide initial conditions that mildly elongate
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molecules for intensity measurements and then increase the field
strength to induce full elongation for length measurement and
relaxation time determination after molecules completely
disengage from the gel. As a non-limiting example, averaging
for 16 frames, or 0.5 seconds, should not be at risk for
significant motion-induced distortion effects. This produces 11
bit data, which provides sufficient resolution for most
intensity determinations; satisfactory results have also been
obtained with only 8 bit data.
To minimize errors arising from relative intensity
measurements, a number of internal standards can be placed into
samples so that fluorescence non-linearities arising from uneven
illumination can be substantially reduced or eliminated. The
internal standard preferably meets several requirements: 1)
negatively charged to avoid sticking to DNA; 2) fluorescent in
the same absorption and emission wavelengths as the DNA-
fluorochrome complex, and 3) readily distinguishable from the
test DNA molecules,'to be measured. Small DNA molecules (20-50
kb) can be used as internal standards. These molecules are
dispersed throughout the sample to be measured and preferably
react identically as the sample DNA to illumination and
electrophoretic conditions (e.g., in free solution). The
internal standards to determine relative mass, instead of using
contiguous restriction digestion products from the same parental
molecule. To optimize these determinations, we will
systematically vary the DNAs used as standards, utilizing
standards of different sizes, and measure their relative
fluorescence intensities. If necessary, empirical correction
factors are calculated to ensure linearity.
Sizing Small DNA Fragments by Measurements of
Fluorescence Intensity: The lower size limit of optical mapping
is currently 30 kb, and it would be useful to extend resolution
to smaller fragnients. Thus, accurately sizing DNA fragments as
small as 500 bp using fluorescence microscopy is an important
step in creating high resolution maps of large DNA molecules,
e.g., using restriction enzymes with 6 bp recognition sites.
Conventional measurements of fluorescence intensity of
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molecules containing small numbers of fluorochromes are plagued
with problems of low signal intensity and low signal/noise
levels. Essentially, when the photon counting level on a
microscope is approached, more than the sample fluoresces and
your microscope is full of stray light. Fluorescence lifetime
capabilities of this instrument can be applied as a
sophisticated filtering system to eliminate stray light and
background fluorescence (87, 88). This approach is expected to
significantly improve the low light detection capability
relative to our present SIT based system.
The heart of the imaging fluorescence lifetime
microscope is the coiled image intensified charge coupled
device, or, simply, ICCD. This low noise device can image under
remarkably low light conditions that approach single photon
counting levels (86). The signal/noise performance is at least
twice as good as a frame averaged SIT camera (89). The ICCD is
also gatable down to 5 ns, which is shorter than most
fluorescent probe lifetimes. The intensification stage on this
camera consists of a microchannel plate, which functions like a
bundle of photomultiplier tubes, so that a small number of
photons triggers an avalanche of electrons that hit a phosphor
screen and produce a bright image. The phosphor screen image
is sensed by a CCD chip attached to the intensifier by a fiber
optic coupler, and the chip-born image is dumped into the camera
controller and digitized (90, 91). Similar devices are often
used for military night vision equipment. As mentioned, the
intensifier is gated so it can be opened and closed, just like a
camera shutter. This "shutter", however, is very fast and has a
gating ratio of greater than 5x106:1. The ICCD is a preferred
imaging system for quantitative work when using fixed, non-
moving samples (92), such as in methods of the present
invention.
To maximize the signal/noise ratio, exploitation of
the gating feature of the ICCD is used to open the shutter only
after the excitation pulse is finished, stray light and
scattering from the illumination source being substantially
eliminated. Hence, having created emission photons exclusively
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from fluorescence under controlled and careful timing of the
image collection, bound from unbound emissions, or stray
fluorescence, can be distinguished on the basis of fluorescence
lifetimes.
As non-limiting example, for the ethidium bromide-DNA
complex, the dye lasers are tuned to 525 nm, and the gate widths
are set to 63 ns, since the lifetime of the bound species is
21.1 ns (93), so approximately 3 t should be optimal. The
lifetime of unbound ethidium bromide fluorescence in water is
only about 1.6 ns, so the free fluorochrome emission will
closely follow the excitation laster profile and are easily
selected against. Other sources of background fluorescence
include immersion oil, glass slides and sample impurities, and
fluorescence from these sources can also be attenuated with this
technique.
Gated pulses can be are timed and synchronized with
fluorescence decay. The gating pulser is timed to produce a
high voltage signal during the fluorescence lifetime of the
fluorochrome-DNA complex. The high voltage pulse opens and
closes the electronic shutter. Illumination are pulsed with a 8
ns FWHM duration so that excitation is present only when the
shutter is closed. Eliminating filters increase light
throughput and remove another source of unwanted fluorescence.
the laser excitation repetition rate is variable (1-100 Hz), and
the fluorescence emissions accumulate as charge on the ICCD
head; a resultant image builds up consisting of bright spots
with intensities proportional to mass.
Two nanosecond lasers are appropriate for these
methods, such as but not limited to, a Continuum Corporation Nd-
YAG pumped TiSaphire tunable solid state laser and a Lambda
Physik excimer pumped dye laser.
The sensitivity and size resolution of such system can
be evaluated using EcoRI digests of lambda bacteriophage DNA
stained with ethidium bromide. Images are generated in the
described system and the spot intensities, corresponding to
single molecules, are tabulated by our image processing
routines. These are subsequently binned to obtain histograms
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depicting intensity populations which correspond to fragment
size populations. This sort of analysis can be done according
to (94) on DNA molecules flowing through a synthetic silicon
matrix. The precision and accuracy of these measurements can be
calculated and used to set proper bin widths for the histogram
analysis.
DNA fragments preferably are in nearly perfect focus.
If fragments are out of focus, intensity values can vary for the
same sized molecule. To ensure that molecules are in focus,
magnesium ions adhered DNA molecules can be used to clean glass
surfaces. Other methods may also include the use of centrifugal
forces to spread DNA fragments in solution or gel out on a glass
surface.
Non-uniform illumination can be corrected by a
combination of careful illumination adjustments and by use of
processing routines developed for relative intensity
measurements in optical mapping. Essentially, this routine
locates local surrounding pixels and uses their intensity values
to calculate local background values. Local background values
will compensate for uneven illumination and thus act as shading
correction.
Other fluorochromes can be used, e.g. those having
varying degrees of sequence specificity and, if appropriate,
fluorochromes with complementary sequence biases used, such as
ethidium homodimer and ethidium-acridine orange heterodimer.
Contrast can be further improved by eliminating unbound
fluorochrome. Ethidium monoazide (Molecular Probes, Inc.) is a
fluorochrome that covalently attaches to DNA in high yield by
photochemical means, and unbound compound can be readily
extracted from the labeled DNA before mounting.
A series of well-defined DNA fragments is added to the
sample as internal fluorescent size standards. The
concentration of fluorescence intensity standards is adjusted so
that they are readily identifiable in any histogram analysis. A
nearly linear relationship between mass and fluorescence
intensity is expected.
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Fluorescence lifetime microscopes can also be used to
improve intensity based sizing for larger fragments (50-1,000
kb) or 1-1,000,000 kb.
The results of the above sizing analysis obtained for
a restriction digest of a pure sample can be an optical
fingerprint and analogous to a fingerprint (without the
hybridization step) derived from gel electrophoretic methods.
Ancillary methods can use this advanced sizing methodology to
produce ordered maps from genomic DNA and YACs of particular
individuals or populations or subpopulations.
Detection Methods for Localization of Specific
Sequences by Hybridization to Single DNA Molecules
While optical mapping creates ordered restriction
maps, these maps alone cannot locate precisely where known
sequences or genes lie on the chromosome. Modern in situ
hybridization techniques (96-99) can locate single copy genes,
and the resolution of localization is steadily improving due to
the trend in using increasingly decondensed chromatin (98, 99).
The chromatin is spread out as much as possible so that the
detected DNA loci are as far apart as feasible. The ultimate
extension of this idea is to use single DNA molecules stretched
to a length corresponding to their molecular contour length and
then fixed. Optical mapping and/or RARE can be used to fine-
map genomic DNA, such as genes in Saccharomyces cerevisae and
Candida albicans or other genes in higher animals, such as
humans.
Gene locations can thus be determined for single,
elongated, fixed molecules by using optical mapping to size
single restriction cuts mediated by RATE, and the sizing
resolution is comparable to that obtained by pulsed field
electrophoresis analysis.
Approaches to precisely locate sequences on large
naked DNA molecules may include techniques of this invention
which allow tagging and detection of RARE-mediated
hybridizations to double-stranded molecules using microscopy.
These new approaches rely, in part, on labeling the RecA-
oligonucleotide filaments prior to complex formation (68) with
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the target DNA. Detection is based on fluorescent beads and on
chemiluminescent tagging using alkaline phosphacase.
Hybridization with tagged RecA filaments to optically mapped
single molecules is similar to in situ Southern analysis.
Providing optical mapping Methods to use RARE for Gene
Localization.
RARE sites can be detected on a large DNA molecule
using optical mapping. However, direct detection of the formed
complex eliminates the requirement for detection of the gap due
to cleavage and serves as a touchstone for the formulation of a
new set of methods for fine mapping known sequences. In direct
detection strategy, tagged RARE binding sites appear along a
molecule as bright spots of a color differing from the main
molecule. The color difference provides, of course, the needed
contrast. This is analogous to in situ hybridization with
different colored probes against DAPI stained chromosomes.
Cleavage Visualized RARE: Specific genes can be
mapped onto single naked DNA molecules using RARE cleavages
coupled with optical mapping. A number important experimental
variables provide optimization, optimizing the RARE reactions to
increase yield of clean fragments, using protocols developed by
Koob and Szybalski (66, 67); (optical contour mapping);
visualizing RARE cleavage in our more advanced microscope
chamber that has electrodes to electrically manipulate and
perturb the system; extending visualization of RATE cleavage to
multiple sites; and combining RARE with other restriction enzyme
digestion during optical mapping. To do this RARE is performed
on a mounted sample, followed by diffusion of another enzyme
into the viewing field, by simply adding enzyme through the same
space used to add magnesium ions for optical mapping.
Attached Fluorescent Beads for Optical recA Mediated
hybridization Detection. Single fluorescent beads are easily
imaged with fluorescence microscopy, including the smallest ones
with a diameter of just 0.01 microns. (Although exceeding the
Rayleigh limit, this bead appears as a bright spot.)
Fluorescent beads are a good way to label single DNA molecules
for image processing because individual beads are intensely
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fluorescent, morphologically distinctive, available in wide
range of fluorochromes of differing spectral qualities, and are
easily attached to oligonucleotides. For example, Molecular
Probes, Inc., sells latex beads with coatings of carboxylate,
avidin or streptavidin in 6 spectral ranges (colors) and sizes
varying from 0.01 to 2 microns. The availability of carboxylate
modified and streptavidin coated beads offers many alternatives
for binding them to DNA molecules.
Synthesizing oligonucleotides can be covalently
attached to a series of differently sized fluorescent beads
(0.01-0.05 microns) to optimize RARE conditions. Smaller beads
are preferable because they diffuse more readily through agarose
gel but larger beads are easier to derivatize due to their
larger surface area (100). Fluorescent beads of similar size
have been imaged electrophoresing through gels by fluorescence
(101). Forming RecA filaments using these modified
oligonucleotides and assaying their formation by functionality
in a RARE test system can also be used. The test system is a 60
base region of the yeast LEU2 gene, and the target LEU2 gene,
located on Chromosome III. The RARE protocol is followed: a
RecA filament is made with this oligonucleotide and diffused
into a chopped yeast gel insert, followed by treatment with
EcoRI methylase, extraction of the targeted complex and
digestion with EcoRI. The digested sample is then run out on a
pulsed field gel for analysis. Conjugating oligonucleotides can
use streptavidin-biotin attachment schemes since biotinylated
oligonucleotides have previously been shown to form functional
filaments with RecA and offer the option of adding beads after
the filament has complexed with the target DNA (102).
Detection of the RecA complex on the microscope can
require visualization of the fluorescent bead attached to the
target molecule,, no addition of enzyme may be needed, no
magnesium ions are diffused in and no prior methylation is
carried out. For microscope work, electrophoretically separated
chromosomal DNAs instead of total genomic material are used.
Samples can consist of our yeast chromosome test system; other
chromosomes with previously mapped sequences can also be used.
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Isolated yeast chromosomal gel bands, are chopped into bead
sized pieces, equilibrated in RARE buffer, melted, and re-
equilibrated at 37 C. Bead-linked RecA filaments are then added
to the molten mixture to form filament complexes at the target
sequence site; the mixing in the molten agarose are more
effective than during diffusion through agarose gel. The molten
RecA-bead-DNA mixture is then stained with DAPI and spread on a
microscope slide for optical mapping. Finally, length and
intensity measurements are used to map the bead position. "Red"
beads (Molecular Probes, Inc.), can be used to provide contrast
to DAPI's blue fluorescence.
Reducing Background from Tagged RecA Filaments: The
efficiency of RecA mediated hybridization is strongly dependent
on the filament size (in bases) and concentration (194). The
amount of labeled RecA filament may be a concern in optically
based methods: too many free fluorescent beaded filaments can
obscure imaging beads present in the complex with target
molecules. The following simple actions can be taken to
eliminate this problem if it occurs:
Carefully titrate the amount of labeled filament and
balance the minimum necessary hybridization efficiency for
convenient observations against contrast quality. RecA-mediated
hybridization does not require the RARE methylation and
restriction enzyme cleavage steps, so that hybridization
efficiencies do not have to be critically optimized for
acceptable results.
Unbound filaments can be diffused out through
dialysis, or mild electrophoresis in gel fixed systems could
selectively sweep filaments from the viewing field and leave the
much larger target-filament complexes in place. If necessary,
additional RecA protein can be added for stabilization.
Providing Chemiluminescent Detection of RecA-Mediated
Hybridization: Chemiluminescent labeling of oligonucleotides for
non-isotopic detection in Southern blots and other techniques is
becoming popular because of its high sensitivity, among other
merits (105). In general, alkaline phosphatase is attached to
oligonucleotides (there are several ways to do this and
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commercially available systems), which are then hybridized to
target DNA. Following formation of hybrids, a chemiluminescent
substrate is added, usually 1,2 dioxetane, which rapidly
decomposes into a chemiluminescence generating compound. Light
is emitted with a maximum at 470 nm and a half life of 2-30
minutes depending upon the chemical environment.
Given the tremendous sensitivity of chemiluminescence
and the availability of high quality commercial kits,
chemiluminescence can be used in this invention to optically
detect RARE on single DNA molecules using the techniques
developed for optical mapping. For example, alkaline
phosphatase can be covalently linked to oligonucleotides (106),
or DNA linked to biotin-streptavidin attachment schemes (107;
with kits commercially available). The conjugated
oligonucleotides will then be made into RecA filaments and
tested for RARE effectiveness as described in the previous
section. One advantage of the biotin-streptavidin mediated
alkaline phosphatase linkage is that excess biotinylated
alkaline phosphatase can be easily dialyzed out of the system to
reduce stray chemiluminescence. A chemiluminescent detection
system can be used with RARE, and optical mapping using most of
the steps described herein. The RecA-oligonucleotide (linked to
alkaline phosphatase)-target DNA complex in molten agarose gel
and then mount this for optical mapping. Instead of diffusing
magnesium ions in to trigger enzymatic cleavage, dioxetane is
diffused, required by the chemiluminescence system, for
visualization of RARE sites. The chemiluminescence activity can
then be visualized through the microscope using an ICCD camera
(105); with no illumination necessary. To image the entire
molecule, DNA-fluorochrome fluorescence can be used, and
different fluorochromes used if intial compounds used quench or
interfere with chemiluminescence.
Using Imaged Energy Transfer to Reduce Background from
Tagged RecA Filaments. An alternative approach is to exploit
energy transfer between the fluorochrome labeled DNA and the
bead attached to the oligonucleotide. Excitation can be
selected making the DNA-fluorochrome complex the donor and the
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bead the acceptor. This would mean that the bead could
fluoresce only when it is within 100 angstroms or less of the
donor. However, efficiency of transfer falls off dramatically
with distance (108). Energy transfer imaging using fluorescence
microscopy with different microscope filter combinations allows
visualization of the donor, acceptor, and the donor-acceptor
pair; these are conveniently slid in and out of the illumination
path. A good energy transfer donor to use here is ethidium
bromide or the homodimer (110), since these fluorochromes bind
tightly the fluorescence yield increases dramatically upon
binding. A concern is that free fluorochrome can act as a
donor, though probably not as effectively the intercalated
material. If free chromophore proves to be problem, the
filament can be split into two parts and fluorescent beads
attached in a head-to-head fashion so that they will serve as
the acceptor-donor pair for energy transfer imaging. Another
concern is that latex beads are notoriously prone to
aggregation, which problem can be solved appropriate selection
and use of chromophores (Molecular Probes, Inc., Portland,
Ore.). Measures ensuring against aggregation include
maintaining some charge on beads through careful attention to
ionic strength, and use of Triton X-100 detergent or BSA.
Increasing Throughput in mapping using Genomic DNA or
YACs. The electrophoretic fingerprinting of YACs is an
important technique in many contig assembly efforts (i, 11-13,
32, 33, 38). Although many gel related manipulations have been
automated (11, 39), the task of running, analyzing and
tabulating the results of thousands gels is far from routine.
One major obstacle in automation is gel electrophoresis. It can
be slow, and there are often difficulties involved in converting
band positions to tabulated numbers automatically and with a
high degree of reliability and accuracy. Manual intervention is
often necessary to check and monitor results. Southern blotting
and hybridization steps are required to complete the
fingerprinting process, and this procedure is also plagued with
difficulties when automated (11, 38-41, 111, 112).
RARE Based Approaches to Mapping.
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Rapid Chromosone Fingerprinting by Optical Mapping, A
Simple Approach: Rapidly fingerprinting of eukaryotic
chromosones, using the fluorescence intensity measurement
methodology described herein can be accomplished by the present
invention without any hybridization. For example, the steps
involved in analyzing YAC fingerprints are described: Yeast
bearing YACs are incorporated into gel inserts (3, 55), and
their DNAs separated using pulsed field electrophoresis in low
gelling temperature agarose. Identified YAC gel bands are cut
out and digested with a series of enzymes, in separate tubes or
together. Mounting and fixation techniques are chosen from the
ones described in this proposal. Preferably magnesium ions are
used to fix small molecules onto the slide after extracting the
DNA from the gel band using standard techniques. Size
distributions are determined from fluorescence intensity
measurements as described herein.
The fingerprints produced by a series of different
restriction enzymes ranging from frequent to rare cutters, are
evaluated to assess contig formation and ordering. YACs are
digested using traditional bulk methods, and their fragments
imaged and analyzed serially on the microscope. In fact, a
robot could also be programmed to produce slides for the
microscope, if desired.
Rapid Production of Ordered Chromosone Restriction
Maps: Since Chromosones have left and right arms, chromosomal
polarity can be established on single molecules by appropriate
hybridization to a distal most unique sequence and imaging the
resulting hybrid (9). Adaption of Smith-Birnsteil partial
digestion analysis (113) can be used to rapidly maps Chromosones
using the techniques of optical mapping.
Optical Mapping of Large Mammalian Genomes: Ferrin
and Camerini-Otero (68) have shown that two RARE sites can be
designed to selectively cleave a sizable mammalian genomic
stretch; it can then be resolved on a pulsed field gel from
uncut genomic DNA. These authors, as well as Koob and Szybalski
(67), suggested that these dissected and isolated genomic
sections could be used to construct locus-specific libraries for
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physical mapping studies. These libraries help in tying contigs
together or in mapping uncltDneable regions, for example.
Although directly mapping the small amount of cleaved, genomic
DNA obtained from gels would be convenient, obviating steps and
artifacts of cloning, the concentration of the recovered DNA is
generally insufficient for direct analysis, except by PCR and
optical mapping.
RARE can be used in this invention to dissect large
regions from mammalian genomes for further analysis by optical
mapping. Human cystic fibrosis (115-117) gene can be used as a
test system, along with the same set of RARE oligonucleotides
described by Ferrin and camerini-Otero (68). Using a series of
well screened STS (118) markers is another approach (67).
However, such analysis are otherwise different. The RARE
products are separated from HeLa cell genomic DNA on a pulsed
field gel and, based on Southern analysis, determination is made
onto where to excise the gel band for optical mapping. The RARE
products are not pure but are highly enriched; ~=heir purity can
be quantitated by Southern hybridization to a series of human
probes. For optical mapping the dissected genomic DNA, a
battery of 6 and 8 base pair recognition restriction enzymes can
be used optical mapping results compared with the detailed,
published ones (115-117). The number of molecules needed to
complete a restriction map will depend on a number of factors,
including the degree of fragment enrichment. RARE-mediated
cleavage of an internal fragment, can also be used as a means of
optically selecting molecules from unwanted ones.
A Flow-Based mapping System. Flow cytometry depends
heavily on flow to accomplish its aims, as the name implies, and
decades of efforts have been invested in perfecting flow systems
to accommodate different types of samples (119, 120). But a
drawback in flow cytometry is that large naked DNA molecules
cannot be routinely sorted, although chromosomes can be. This
is understandable since large DNAs are very easily broken by
shear forces generated in typical flow cytometry
instrumentation. Gentle solvent flow fields can be used
(e.g.,5x10-z nl/sec at 100x20 micron opening) to move large DNA
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molecules in fluid without any apparent breakage, as discerned
by fluorescence microscopy. Solvent flow fields with low rates
of shear (shear is the change of solvent velocity with distance
across a flow) can be exploited to stretch out and align DNA
molecules as big as those contained within mammalian chromosomes
(61, 83, 85, 121). Viscoelastic measurement (61, 83, 122) of
chromosomally sized DNA molecules rely on flow fields to stretch
out long molecules for subsequent relaxation measurement.
Solvent flow has also been used by Smith and Bustamante to
stretch out tethered lambda bacteriophage DNA concatamers (77).
In addition, Yanagida and colleagues used solvent flow to
elongate molecules in their pioneering DNA imaging work (44,
81). Gentle flow fields do not break large DNA molecules.
A microscope mounted chamber can be used to
simultaneously flow and elongate large DNA molecules, with the
following objectives in mind: 1) evaluation of flow chamber
characteristics and their effect on DNA stability and
elongation, with respect to imaging, 2) control of molecular
elongation with flow rate, 3) development of systems to deliver
reagents and enzymes to flowing molecules, with concurrent
observation of molecular events; adapting our software to image
and size flowed molecules; creation of high speed restriction
maps using this system.
Flow Chamber Design Fluid: Fluid flow fields and
electrical fields have somewhat complementary engineering
needs,k so that chambers designed utilizing both effects should
be robust and flexible. The chamber is designed to liberate DNA
molecules embedded in gel; these samples can be gel inserts or
excised electrophoresis gel bands. DNA molecules are moved from
the gel into a thin space for observation. The flow field
accomplishes two operations simultaneously. It moves DNA
molecules out of the "extraction area" into the viewing area,
and it elongates and positions large DNA molecules in the flow
streamlines for optical measurement.
Phased Restriction Maps from Flowed Molecules: To
perform optical mapping restriction enzyme or magnesium ions are
used to trigger digestion into the flow chamber after the
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molecule is aligned and elongated in the field to induce
cleavage. Controlling these steps is important. The
restriction enzyme must be triggered, for example, by the
addition of magnesium ions, and then its cutting imaged before
the resultant fragments have flowed out of view. Additionally,
restriction fragments should retain their relative positions to
remain phased. Avoidance of turbulence is important, and the
literature is plentiful on flow cytometry techniques to handle
such difficulties. If necessary, chambers cam be constructed to
produce an accelerating flow rate (easily accomplished with a
tapered geometry) so that fragments will separate efficiently
when cleaved. Fragment sizes are determined using known and
described methods. Large fragments (e.g., >30 kb) are sized
using fluorescence intensity ratio, apparent and absolute
length, and relaxation measurements performed at flow cessation.
Small fragments (e.g., <30 kb) can be sized using the intensity
methods described earlier for the gel-fluid interface system and
the fluorescence lifetime instrumentation if great sensitivity
is needed. Compromises may be made here concerning flow rate,
illumination intensity and imaging time, which affects the
number of usable gray levels (bit depth).
A less demanding measurement is determining size
distributions of restriction digests performed in a test tube.
Full digests can yield fingerprints, while partial digests with
end-labeled molecules (using beads,) can produce fully ordered
maps. If the flowed "in situ restriction digest" disorders
fragments, the map defaults to an unordered, but still useful
fingerprint.
Rapid Maps for Human Genome(s): A main purpose in
providing a flow based restriction mapping system is speed and
simplified automation of analysis techniques as compared to gel
based approaches. The throughput of the flow system described
herein can image 5-500 kb molecules/minute; or additionally 5-10
molecules can be imaged in parallel, e.g., 25-5000 kb
molecules/minute. In 1 hour, 150 mgb can be processed, which is
equivalent to 1.5 human chromosomes. In 24 hours one human
genome worth of DNA can be imaged. These numbers compare
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favorably with the Cohen laboratory YAC fingerprinting
throughput of 61 YACs/day/person (11); and this rate only
describes the film to computer file stage. The potential high
throughput capabilities of the optically based system described
here, coupled with its high information content, provides such
rapid mapping:
If 500 kb molecules are digested while flowing to
produce ordered restriction maps could be made from these
fragments with a high degree of confidence, depending upon the
number of cuts made per 500 kb fragment and the precision of
sizing. Obviously more cuts per 500 kb fragment simply contig
formation. The Smith-Birnsteil partial digestion approach
discussed previously can also be used in here if there are
problems with the flow-and-cut approach. Briefly stated,
ordered restriction maps would be invaluable for dependably
forming mapped (or fingerprinted) YACs into contigs and
determining internal order. Ordered, accurate restriction maps
of 500 kb fragment might reduce the number of genome equivalents
needed for coverage, due to less overlap needed to for contig
assignment. Additionally, these maps are an important resource
for any studies concerning genome structure and organization. A
good source of genomic DNA might be from flow sorted chromosomes
that are partially digested to produce 500 kb overlapping
fragments. Here again optical mapping should ideally interface
with this relatively meager (in terms of number of DNA
molecules) source of DNA. With enough precisely sized fragments
generated from a single molecule it is possible to construct
contigs without hybridization-fingerprinting because optical
mapping can produce data with greater information density than
electrophoresis based methods.
In comparison, electrophoretic methods poorly
characterize size distributions and requires hybridizations to
decompose gel bands consisting of multiple fraginent types. it
is also not common to run agarose gels and discern 100 separate
bands with any clarity. This approach is suitable for whole
genome analysis in a global approach, analogous to the Cohen
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laboratory's largely successful tact in using fingerprinted
large YACs.
Ordered restriction maps provide more information than
simple fingerprints so that less overlap may be necessary for
contig formation (124) than simple unordered restriction
fingerprints (125). However, these 500 kb partial digestion
fragments can also be fingerprinted more simply by digesting
them to produce unordered or partially ordered maps. If
necessary such fingerprints could also be augmented with RecA
mediated hybridization of sequences such as LINE-1 (11, 126) in
the same way these commonly used on Souther blots (11, 38;).
In summary, the medium-based sizing process of this
invention involves characterization of molecules using a
microscope. Molecules, particularly small or medium-sized
molecules, are placed in a medium and mounted on a slide using
conventional techniques. Large molecules are mounted on a
microscope slide using spermine condensation, which avoids
breakage problems. At some point the molecules may be stained.
The molecules may be perturbed in the medium by the application
of an electrical field. The field is then shut off, allowing
the molecules to relax to their equilibrium conformation, which
on the average is spherical or ellipsoidal, or to assume a
certain position. An image processor connected to a video
camera counts the molecules and follows their shape changes.
The kinetics of relaxation, reorientation and rotation of the
molecules, as well as their length and diameter are calculated
automatically, and molecular weights for all of the imaged
molecules are calculated from established relationships.
The following examples are offered in order to more
fully illustrate the invention, but are not to be construed as
limiting the scope thereof.
EXAMPLE 1. Preparing DNA for Microscopy
G bacteria was grown as described by Fangman, W.L.,
Nucl. Acids Res., 5, 653-665 (1978), and DNA was prepared by
lysing the intact virus in 1/2 X TBE buffer (lX: 85 mM Trizma
Base (Sigma Chemical Co., St. Louis MO), 89 mM boric acid and
2.5 mM disodium EDTA) followed by ethanol precipitation; this
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step did not shear the DNA as judged by pulsed electrophoresis
and microscopic analysis.
DNA solutions (0.1 microgram/microliter in 1/2 X TBE)
were diluted (approximately 0.1-0.2 nanogram/al agarose) with
1.0%- low gelling temperature agarose (Sea Plague, FMC Corp.,
Rockport ME) in 1/2 X TBE, 0.3 micrograms/ml DAPI (Sigma
Chemical Co.), 1.0t 2-mercaptoethanol and held at 65 C. All
materials except the DNA were passed through a 0.2 micron filter
to reduce fluorescent debris. Any possible DNA melting due to
experimental conditions was checked using pulsed electrophoresis
analysis and found not to be a problem.
EXAMPLE 2. Imaging DNA in a Gel
The sample of Example 1 was placed on a microscope
slide. To mount the sample, approximately 3 microliters of the
DNA-agarose mixture were carefully transferred to a preheated
slide and cover slip.using a pipetteman and pipette tips with
the ends cut off to reduce Shear. Prepared slides were placed
in a miniature pulsed electrophoresis apparatus as shown in
Figures 1 and 2. All remaining steps were performed at room
temperature. Samples were pre-electrophoresed for a few minutes
and allowed to relax before any data was collected. Pulsed
fields were created with either a chrontrol time (Chrontrol
Corp., San diego, CA) or an Adtron data generating board (Adtron
Corp., Gilbert, AZ) housed in an IBM AT computer and powered by
a Hewlett Packard 6115A precision power supply. Field Strength
was measured with auxiliary electrodes connected to a Fluke
digital multimeter (J. Fluke Co., Everett, WA). A Zeiss
Axioplan microscope (Carl Zeiss, West Germany) equipped with
epifluorescence optics suitable for DAPI fluorescence and a
Zeiss 100x Plan Neofluar oil immersion objective was used for
visualizing samples. Excitation light was attenuated using
neutral density filters to avoid photodamage to the
fluorescently labeled DNA. A C2400 silicon intensified target
(SIT) camera (Hamamatsu Corp., Middlesex, NJ) was used in
conjunction with an IC-1 image processing system (Inovision
Corp., Research Triangle Park, NC) to obtain and process video
images from the microscope. Images were obtained continuously
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at the rate of one every five or six seconds, and as many as 200
digitized images could be stored per time course. Each
digitized time-lapse image benefitted from the integration of 8
frames obtained at 30 Hz, which was fast enough to avoid
streaking due to coil motion. After the time-lapse acquisition
was complete, the microscope was brought out-of-focus and a
background image was obtained. Each time-lapse image was
processed by first attenuating a copy of the background image,
so that the average background intensity was 82%- of the average
time-lapse image intensity. The attenuated background was
subtracted from the timelapse image and the resultant image was
then subjected to a linear-stretch contrast enhancement algo-
rithm. Photographs of the processed images were obtained using
a Polaroid Freeze Frame video image recorder (Polaroid Corp.,
Cambridge, MA).
EXAMPLE 3. Perturbing Molecules in a Gel
The molecules of Example 2 were perturbed by POE. POE
was accomplished by using a series of relatively short normal
pulses of a chosen ratio and then after a longer time period,
the polarity of one of the fields was switched. The switch time
and normal field ratio are analogous to the pulsed
electrophoresis variables of pulse time and field angle.
The nomenclature used to describe a POE experiment is
as follows: 3,5-80 second pulses, 3 volts/cM). "3,5-80
seconds" means a 3 second pulse south-north, followed by a 5
second pulse east-west; after 80 seconds of this 3,5 second
cycle, the polarity of the 5 second pulse is changed (west-east)
for another 80 seconds, and a zig-zag staircase path is defined
for the molecules involved. The pulse intensity was 3 volts/cM.
In this Example, epifluorescence microscopy was coupled with the
POE method to enable the general study of DNA conformational and
positional changes during electrophoresis. While the POE method
using the adapted microscopy chamber shown in Figure 2 was used
in this experiment, ordinary electric fields switched on and off
could have been used. POE offers certain advantages when
electric fields are to be applied at different angles, as may be
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needed to rotate a molecule about its long axis. Figures 1 and
2 show diagrams of the adapted POE chamber.
EXAMPLE 4. Observing and Measuring
Molecular Relaxation in a Gel
The relaxation of the G bacteriophage DNA of Examples
1-3 was observed after POE was conducted for 600 seconds (3,5-80
second pulses, 3 volts/cm).
The image processor is used to quantify and automate
the imaging of the relaxation process, for example, through
"feature analysis". Feature analysis works after successive
images have been digitized and stored, as shown in Fig. 3(a).
The image processor then identifies discrete objects in the
images, numbers them, and characterizes them according to shape.
For example, the computer determines the effective ellipsoid
axes (long and short) for a collection of distorted coils and
calculate these features as a function of time as the coil
approaches a spherical conformation during the relaxation
process. Other types of computerized measurements also can be
made to characterize the DNA.
The images displayed in Fig. 5, obtained at 12 second
intervals, show the relaxation of several molecules over a 96
second time span. In (a), several coils are shown 3 seconds
after the applied field was turned off. The coils appear to
relax through the same corrugated staircase path defined by the
applied electrical pulses (see molecules marked by arrows) as
determined by the limits of microscopic resolution. In (c), a
molecule is shown splitting into two, and by (j), all coils have
relaxed to a round, unelongated conformation. The bar shown in
(j) is 10 microns in length.
EXAMPLE 5. Determining the Molecular Weight of One or More
Molecules by Measuring Relaxation Kinetics
Molecules of known molecular weight are prepared for
imaging according to the procedures of Examples 1-3, and the
relaxation time of the molecules is determined by the methods of
Examples 1-4. Relaxation time.data is collected by imaging and
is used to calculate a mathematical relationship between
molecular weight and relaxation time of DNA molecules of similar
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composition. The relaxation time of a sample of molecules of
unknown size is then measured, and the size of the molecules is
calculated using the mathematical relationship determined on the
basis of molecules of known size.
EXAMPLE 6.
Determining the Molecular Weight of One or More
Molecules by Measuring Reorientation
Rate in a Gel
Polymers of any size, but particularly those that are
too small to image (less than approximately 0.1 micron), are
sized in a matrix such as agarose or polyacrylamide gel by
measuring the reorientation rate as induced by an applied
electrical field. Although a reorientation measurements could
be done in free solution, a matrix is preferred because it
prevents unnecessary polymer convection and movement. Addition-
ally the presence of a matrix may enhance the size sensitivity,
partly because the orientation mechanism is different. POE is
particularly useful for measuring reorientation time because of
its experimental versatility and very high size resolution of
perhaps 15 to 20 megabases. Stiff polymers such as DNA
molecules (sized less than 150 base pairs) exist in solution as
rods and the rotational diffusion coefficient (the friction felt
by the rod as you try to spin about its long axis) varies as M3.
Using microscopy, molecules which are large enough to be imaged
are visualized, and their reorientation time is determined from
the images. For any size of molecules, particularly those which
are too small to visualize, the reorientation time of each rod
in the field of view is preferably measured by spectroscopic
methods. Two such methods are described in detail below, namely
fluorescence dichroism and birefringence:
1) A chromophore that binds in a sterically predict-
able way (ethidium bromide intercalates into DNA molecules) is
attached to a polymer molecule. Polarized radiation is used to
excite the chromophore. Measuring the total fluorescence
intensity temporally provides orientation information of each
molecule. The fluorescence radiation of each molecule in the
microscope field is measured using a sensitive micro-channel
plate detector.
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2) The orientational dynamics of a molecule is
followed with birefringence measurements. Birefringence
techniques measure the change of refractive index, which is
easily correlated with the orientation of macromolecules in
solution or in a matrix. Birefringence measurements are taken
while the DNA molecules are undergoing gel electrophoresis.
When an electrical field is applied, the DNA molecules stretch
out and align with the field, thereby changing the refractive
index. By measuring the change of birefringence with time, it
is possible to understand details of DNA blob train motion as
- the molecule orients with the applied electrical field.
More specifically, birefringence measurements are made
by determining the phase difference of two orthogonally
polarized planes of laser radiation (red light) differing by a
small frequency difference (supplied by the two frequency
laser). As the molecules align with the applied electrical
field (in the POE chamber 74), which is generated by pulse
controller 82, the refractive index changes with molecular
alignment. Light is detected by detector 76, and results in a
phase difference in the transmitted radiation, which is measured
by the phase detector 78 (Fig. 3(b)) by comparing the value to a
standard, sourced at laser 70. The phase difference data
obtained as a function of time (the period of field application)
is digitized and stored on computer 80 for later retrieval and
analysis.
The instrument depicted in Figs. 1 and 2 applies the
necessary fields to cause molecular reorientation. Many
different rotational schemes can be described to optimally size
molecules in the field. For example, the rotating field
frequency can be swept to find resonant frequencies with the
polymer sample.
E]LAMPL$ 7
Determining the Molecular Weight of One or More
DNA Molecules by Measuring the Rotation Time
of the Molecules in a Gel
Molecules in the shape of rods or stiff coils are
prepared and observed as in Examples 1-4, except that an
acrylamide, rather than agarose gel optionally may be used.
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The rate of rotation of a coil or a rod is measured
with a microscope-based system using any one of the techniques
described above in Example 6. Measurements are made of a
sinusoidally varying signal as the molecule spins about its
center. The sinusoidal signal is used to determine the polymer
size or molecular weight by fitting the period of the sinusoidal
signal to the rotational frictional coefficient, which varies as
the cube power of the rod length. In other words, the measured
angular velocity as measured from the sinusoidal signal
(radians/sec.) varies as the rod length cubed in free solution
(Boersma, S. (1960) J. Chem Phys. 32: 1626-1631, 1632-1635).
The conditions for a proposed series of experimental
runs, with constant t, are shown below.
M E At 6;
MolecularSize ElectricField Duration of Incremental
(base prs or Strength each Pulse angle (in
kilo bases) (volt/cm) (Sec) clockwise
direction
(Deg.)
50 bp 5 1X104 10
150 bp 5 1X10-4 10
50 kb 5 1 10
500 kb 5 5 10
500 kb 5 900 10
Thus, in the first example, pairs, triplets or other
sets of pulses of 5 volts/cm are successively applied for .1
millisecond in opposite directions, with the direction of the
first of each successive set of pulses increasing by 10 degrees
in a clockwise direction away from the starting point.
Molecules of known molecular weight are placed in a
gel, and their rotation rate is determined when the above-
described electric fields are applied. Rotation time data is
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collected and is used to calculate a mathematical relationship
between molecular weight and rotation time of G bacteriophage
DNA molecules in a particular gel. The rotation time of
molecules of unknown size is then measured, preferably using a
similar electric field, and the size of the molecules is
calculated using the mathematical relationship determined on the
basis of molecules of known size.
EXAMPLE 8
Determining the Molecular Weight of One or More
Molecules by Measuring Curvilinear Length of DNA
Molecules in a Gel
The procedure of Examples 1-4 is followed for
molecules of known molecular weight. Measurements of the
curvilinear length of the molecules while they are in a
perturbed state is collected by visualizing the molecules and is
used to calculate a mathematical relationship between molecular
weight and length. The curvilinear length of perturbed
molecules of similar composition and unknown size is then
measured using the procedures of Examples 1-4, and the size of
the molecules is calculated using the mathematical relationship
determined on the basis of molecules of known size. Figs. 4 and
5 show perturbed molecules for which curvilinear length
measurements can be made.
EXAMPLE 9
Determining the Molecular Weight of One or More
Molecules by Measuring Diameter of DNA
Molecules in a Gel
The procedure of Examples 1-4 is followed for
molecules of known molecular weight, except that measurements
are made when the molecules are in a completely relaxed state.
Measurements of the diameter or diameters of the substantially
spherical or ellipsoidal G bacteriophage DNA molecules are
collected and are used to calculate a mathematical relationship
between molecular weight and diameter of G bacteriophage DNA
molecules in the gel. The diameter of molecules of unknown size
is then measured, and the size of the molecules is calculated
using the mathematical relationship determined on the basis of
molecules of known size. Figs. 4(a) and 5(j) show relaxed
molecules for which diameter measurements can be made.
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EXAMPLE 10
Preparing Large DNA Molecules for Imaging
Chromosomal DNA molecules from Saccharomvces
cerevisiae were prepared and isolated using the insert method
and pulsed electrophoresis. Low gelling temperature agarose gel
(FMC Corp. Rockland Maine) was used for preparation to permit
relatively low temperature melting. Since W radiation can
break DNA molecules, desired bands were cut out of the gel,
guided by ethidium stained flanking edge sections that were cut
out of the gel, guided by ethidium stained flanking edge
sections that were cut out of the gel, which were then
photographed on a 301 nm transilluminator apparatus. The bands
were then weighed and equilibrated with a 10-fold excess of 10mM
spermine in water for 3 hours at room temperature. Spermine
requires a very low ionic strength environment to condense DNA
and, fortunately, the buffers used in electrophoresis are low
ionic strength, thus eliminating the need for an equilibration
step. The equilibrated samples were then melted in an oven at
74 C for two hours and after melting. DAPI (1 microgram/ml) and
2-mercaptoethanol (l%-) were added. 3 microliters of the melted
agarose/DNA mixture were carefully applied to a pre-heated
microscope slide and a cover slip was placed on top before the
mixture gelled. The slide was then viewed using a Zeiss
Axioplan epifluorescence microscope fitted with a 100X Plan
Neofluar objective and showed small intensely bright balls which
could be decondensed by the addition of salt, through the edges
of the coverslip sandwich.
As mentioned above, spermine is particularly useful in
an environment of low ionic strength. On the other hand, if DNA
molecules are placed in a highly ionic environment, the same
type of condensation effect are accomplished with alcohol.
Neither of these examples are to be construed as limiting the
scope of the invention.
EXAMPLE 11
Restriction mapping Schizosaccharomyces
pombe Chromosomal DNA Molecules
The DNA of Schizosaccharomyces pombe, a fungus with a
genome size of about 17-19 megabases distributed on three
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chromosomes 3, 6 and 8-10 megabases in size, is prepared for
microscopy by condensation and uncollapsing, according to the
method of Example 10. The 3-5 microliter agarose mixture
contains approximately 0.1 nanograms of DNA, 0.5% b-
mercaptoethanol, 1 microgram/ml DAPI, 100 micrograms/ml bovine
serum albumin (acetylated; Bethesda Research Laboratories,
Gaithersburg, MD) and 10-20 units of an appropriate restriction
enzyme. This mixture is briefly held at 37 C and carefully
deposited on a microscope slide and then topped with a
coverslip. Prior to digestion with restriction enzymes the DNA
is stretched by one of two ways: (1) the liquid
siide/agarose/coverslip sandwich is optionally sheared slightly
by moving the coverslip or (2) an electrical field is applied
using, for example, the POE instrument described in Fig. 3. A
10 mM magnesium chloride solution is then diffused into the
sandwich once the gel has set. When the magnesium ions reach
the DNA/enzyme complex, the enzyme cleaves the DNA molecule.
The positions of the restriction cutting sites are
determined by following the DNA strand from one end to the other
using the microscope setup and noting cut sites. These sites
appear as gaps in the strand, which is continuous before
enzymatic digestion. The size of each of the fragments is then
determined by the microscopic methods of this invention,
including, (1) measuring the curvilinear length of each
fragment, (2) allowing the fragments to relax and measuring
their diameter, (3) perturbing the conformation of each fragment
with an applied electrical field or flow field (as generated by
moving solvent through a gel) and measuring the relaxation
kinetics with direct visual detection of conformational and
positional changes or microscopy combined with spectroscopy.
Direct visual observation is preferred for larger molecules,
while the other methods are well suited for fragments too small
to image.
The resulting sample when viewed using a fluorescence
microscope shows a number of bright balls of three different
sizes, with diameters varying as M.33, which is based upon the
formula for the volume of a sphere, 4/3R3. The gel also contains
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a restriction enzyme which is active only when magnesium ions
are present.
EXAMPLE 12
In situ Hybridization of Nucleic Acid Probes to
Single DNA Molecules
Nucleic acids are prepared for microscopy as described
in Examples 1-4 above. The agarose medium containing the
nucleic acid molecules also contains labelled probes and a
recombinational enzyme, recA, which mediates strand displacement
of the target molecule by the probe. Strand displacement and
pairing occurs by D-looping (see Radding, C., Ann.Rev.Genet.
16:405-37 (1982)). ATP and magnesium ions are added to begin
the reactions. These ingredients are diffused into the
slide/gel/ coverslip sandwich as described in Example 11. The
reaction is incubated at 37 C. Many different target molecules
are simultaneously analyzed, using probes with different labels.
Variations of the method of this invention other than
those specifically described above are within the scope of the
invention. For example, other parameters of the molecules can
be measured, and various type of microscopes and spectroscopic
equipment may be used. The pulsing routines for effecting
molecule rotation can be varied. Combinations of the above-
described techniques are also contemplated. For example,
combinations of various types of external forces, mediums and
spectroscopic techniques are within the scope of the invention.
Furthermore, a measuring technique may be repeated several
times, and the measurements from each trial may be averaged.
EXAMPLE 13
Ordered Restriction Maps of Saccharomyces Cerevisiae Chromosomes
Constructed by optical mapping
Optical mapping (e.g, as shown in figure 6), images
are made stained, single, deproteinized DNA molecules during
restriction enzyme digestion, allowing direct, ordered mapping
of restriction sites. In brief, a flow field (or in principle,
or other kinds of electrical field) is used to elongate DNA
molecules dissolved in molten agarose and fix them in place
during gelation.
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As a non-limiting example, yeast chromosomal DNA
(yeast strain AB972) was resolved by pulsed electrophoresis
(Schwartz et al., Cell 37:67 (1984)) using 1.00t Seakem low
melting agarose (FMC), 1/2x TBE(42.5mM Trizma base, 44.5mM boric
acid, 1.25mM disodium EDTA). Cut gel bands were repeatedly
equilibrated in TE (10mM Tris-Cl, 1mM EDTA, pH8.0). The gel
embedded, purified chromosomes were then equilibrated overnight
at 4 C in magnesium-free restriction buffer containing 0.1 mg/ml
acetylated bovine serum albumin, 1t 0-mercaptoethanol, 0.1t
Triton X-100 (Boehringer Manheim, membrane quality), and 0.2
ug/ml 4', 6-diamino-2 phenylindole dihydrochloride (DAPI) with
slow shaking. Equilibrated samples ranging in volume from 50 to
100 ul were melted at 72 C for 5 minutes, and then cooled to
37 C. Approximately 0.3 - 0.5ul of enzyme (2 to 14 units/ l)
was spread on a slide. Enzyme reaction temperatures were as
recommended by manufacturers. /3-mercaptoethanol was added to
discourage photolysis M. Yanagida et al. in Applications of
Fluorescence in the Biomedical Sciences, D.L. Taylor et al.,
Eds. (Alan R. Liss, New York, 1986), pp. 321-345. and was tested
at this concentration for any deleterious effects on digestion
using electrophoresis. A 7 l volume of the melted sample was
typically pipetted (slowly) using a wide bore pipette tip onto
an 18X18 mm cover glass and rapidly deposited onto a slide.
Timing and quenching of the gel is critical for controlling
elongation. The reaction chamber was then sealed with mineral
oil to avoid evaporation, and the agarose was allowed to gel for
at least 30 minutes at 4 C, prior to diffusion of 50mM MgC12
through an open space. For chromosome I(240kb) and III (345kb),
slides were in a cold desiccator (4 ) prior to casting to hasten
gelling avoiding premature molecular relaxation. For the larger
chromosomes, which relax more slowly, slides were kept at room
temperature. The slide was placed on a temperature controlled
microscope stage at 37 C (except CspI, 30 C). The gelatin
process restrains elongated molecules from appreciably relaxing
to a random coil conformation during enzymatic cleavage. A
restriction enzyme is added to the molten agarose-DNA mixture
and cutting is triggered by magnesium ions diffused into the
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gelled mixture (mounted on a microscope slide). Cleavage sites
are visualized as growing gaps in imaged molecules. DNA
molecules were imaged using a Zeiss Axioplan or Axiovert 135
microscope equipped for epi-fluorescence (487901 filter pack for
UV excitation and Blue emission) and a 100X or 63X Plan-Neofluar
objective (Zeiss) coupled to Hammamatsu C2400 SIT cameras. Care
was taken to adjust the camera controls to avoid saturating the
digitizer at either end of the intensity range. Every 20
seconds, 32 video frames were digitized to 8 bits and integrated
to give 13 bit precision by a Macintosh based Biovision image
processor or a Pixel pipeline digitizer (Perceptics Corp.). A
computer controlled shutter was used to limit illumination to
1.5 seconds per image giving a total of about 135 to 255 seconds
for typical experiments. Neutral density filters were used to
keep the illumination intensity below 100 W measured at the
objective. Control experiments showed no damage to DNA
molecules under these conditions. Digitized images were
recorded directly to disk and archived on tape. The resulting
fragments are sized in two ways: by measuring the relative
fluorescence intensities of the products, and by measuring the
relative apparent DNA molecular lengths in the fixating gel.
Maps are subsequently constructed by simply recording the order
of the sized fragments. Length and relative fluorescence
intensity were calculated to 16-bit precision using a modified
version of NIH Image for Macintosh by Wayne Rasband, available
upon request from the authors (e-mail huff@ mcclb0.med.nyu.edu).
Briefly, the original unprocessed image was displayed in an
enlarged format and an overlay image was prepared by manually
tracing the DNA. The length map was made directly from this
overly. For intensity calculations, the 13-bit raw data image
was smoothed and the overlay image was dilated five times to
cover all foreground pixels. For each pixel marked on the
overlay, a synthetic background value was calculated as the
weighted average of surrounding pixels, with a weight that
decreased with distance, but was zero for all marked pixels.
These values are intended to approximate those which would have
been measured had the DNA been absent. The intensity of a
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particular DNA fragment was the sum of all pixels of the
fragment minus the matching background pixels. The are of the
fragment was the original overlay dilated twice. This process
was repeated for each frame of raw data which had an overlay
image, excluding those with poor focus. Intensity results were
averaged for five images following a cut, and the relative sizes
of the two fragments were calculated as x/(x+y) and y/(x+y). If
fragment y later cuts into u and v, then (y/(x+y))(u(u+v)) is
used for the size of u. The resulting numbers constitute a
single sample for the purposes of subsequent analysis.
Averaging a small number of molecules rather than utilizing only
one improves accuracy and permits rejection of unwanted
molecules. The samples were averaged and the 90$ confidence
interval on the mean was calculated using the t distribution
with n-i d.f. and the sample standard deviation. This
calculation is valid if the data represent random samples from a
normal distribution. There is a 90t chance that the population
mean falls within the confidence interval. For chromosome I,
the reported confidence interval was found by taking the lower
bound from the short fragments and upper bound from the long
fragments. The 90% confidence interval for the population
standard deviation was calculated using the sample standard
deviation, the number of samples, and the chi-square
distribution with n-1 d.f. The midpoint of this interval was
used to estimate the population standard deviation. The
coefficient of variation (CV) is the estimated population
standard deviation divided by the sample mean. The pooled
standard deviation is the square root of the average of the
variances. The relative error is the differences between our
value and the reported value divided by the reported value.
Optical map production is very rapid because of the combination
of restriction fragment ordering in real time with fast accurate
sizing techniques. Optical mapping is a powerfsl new technology
for rapidly creating ordered restriction maps of eucaryotic
chromosomes or YACs, without the need for analytical electropho-
resis, cloned libraries, probes, or PCR primers. Incremental
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technical improvements should enable the rapid high resolution
mapping of mammalian chromosomes and ordering of YACs.
Gel fixation and mechanics of DNA relation under
tension and cleavage. A single large DNA molecule 200 gm long
(600 kb) is a random coil in solution which can be visualized as
a loosely packed ball averaging 8 m across (Roberts, 1975).
Optical mapping begins with stretching out such a DNA molecule
and fixing it in place to inhibit rapid relation, prior to
imaging by light microscopy. The fixed molecule must lie within
a shallow plane of focus for successful imaging. Elongated
molecules in a gel behave mechanically like a stretched spring
(Schwartz, Koval, 1989): fixed molecules are under tension
which is released during coil relaxation to a random
conformation. However, excess fixation is undesirable for
optical mapping, since restriction cleavage sites must relax to
be detected and imaged as growing gaps.
Zimm (Zimm, 1991) has modeled DNA molecules embedded
in agarose gel, during electrophoresis, as a series of connected
pools of coil segments under tension with each other, and
calculates that the force (fi) associated with the free energy
change of shuttling coil segments between pools is given by
fi=3kT/(2nib)((a2/nib2)-1)+(kT/b)InC, (Chumakov, Nature 359,380
1992) where k is the Boltzmann constant, a is the gel pore
diameter, ni is the number of associated coil segments, b is the
coil segment length, T is the temperature and C is a constant
relating to coil segment structure. This result shows that the
tension developed between pools is inversely related to the
number of segments contained with a pore volume (Eq.1). It
follows that a stretched our, elongated molecule is under more
tension than a compact, relaxed one.
Large DNA molecules can be stretched out in molten
agarose by flow forces and then rapidly fixed in place by
agarose gelation, without application of electrical fields.
Yeast chromosomal DNA (yeast strain AB972) was resolved by
pulsed electrophoresis (D. C. Schwartz and C.R. Cantor, Cell
37,67 (1984)) using 1.00% Seakem low melting agarose (FMC), 1/2x
TBE(42.5mM Trizma base, 44.5mM boric acid, 1.25mM disodium
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EDTA). Cut gel bands were repeatedly equilibrated in TE (10mM
Tris-Cl, 1mM EDTA, pH8.0). The gel embedded, purified
chromosomes were then equilibrated overnight at 4 C in
magnesium-free restriction buffer containing 0.1 mg/ml
acetylated bovine serum albumin, it fl-mercaptoethanol, 0.1t
Triton X-100 (Boehringer Manheim, membrane quality), and 0.2
ug/ml 41, 6-diamino-2 phenylindole dihydrochioride (DAPI) with
slow shaking. Equilibrated samples ranging in volume from 50 to
100 ul were melted at 72 C for 5 minutes, and then cooled to
37 C. Approximately 0.3 - 0.5u1 of enzyme (2 to 14 units/ l)
was spread on a slide. Enzyme reaction temperatures were as
recommended by manufacturers. fl-mercaptoethanol was added to
discourage photolysis M. Yanagida et al. in Applications of
Fluorescence in the Biomedical Sciences, D.L. Taylor et al.,
Eds. (Alan R. Liss, New York, 1986), pp. 321-345. and was tested
at this concentration for any deleterious effects on digestion
using electrophoresis. A 7 1 volume of the melted sample was
typically pipetted (slowly) using a wide bore pipette tip onto
an 18X18 mm cover glass and rapidly deposited onto a slide.
Timing and quenching of the gel is critical for controlling
elongation. The reaction chamber was then sealed with mineral
oil to avoid evaporation, and the agarose was allowed to gel for
at least 30 minutes at 4 C, prior to diffusion of 50mM MgC12
through an open space. For chromosome I(240kb) and III (345kb),
slides were in a cold desiccator (4 ) prior to casting to hasten
gelling avoiding premature molecular relaxation. For the larger
chromosomes, which relax more slowly, slides were kept at room
temperature. The slide was placed on a temperature controlled
microscope stage at 37 C (except CspI, 30 C). Experimentally,
the kinetics of gelation are controlled by temperature, and
optimization of the annealing conditions. For our analysis, DNA
coils must be critically stretched: too much and molecule
becomes difficult to image; too little, and there is
insufficient tension to reveal cut sites. Yeast chromosomal DNA
(yeast strain AB972) was resolved by pulsed electrophoresis (D.
C. Schwartz and C.R. Cantor, Cell 37,67 (1984)) using 1.00t
Seakem low melting agarose (FMC), 1/2x TBE(42.5mM Trizma base,
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44.5mM boric acid, 1.25mM disodium EDTA). Cut gel bands were
repeatedly equilibrated in TE (10mM Tris-Cl, 1mM EDTA, pH8.0).
The gel embedded, purified chromosomes were then equilibrated
overnight at 4 C in magnesium-free restriction buffer containing
0.1 mg/ml acetylated bovine serum albumin, 1t ~-mercaptoethanol,
0.1g Triton X-100 (Boehringer Manheim, membrane quality), and
0.2 ug/mi 4', 6-diamino-2 phenylindole dihydrochloride (DAPI)
with slow shaking. Equilibrated samples ranging in volume from
50 to 100 ul were melted at 72 C for 5 minutes, and then cooled
to 37 C. Approximately 0.3 - 0.5ul of enzyme (2 to 14 units/ l)
was spread on a slide. Enzyme reaction temperatures were as
recommended by manufacturers. /3-mercaptoethanol was added to
discourage photolysis M. Yanagida et al. in Applications of
Fluorescence in the Biomedical Sciences, D.L. Taylor et al.,
Eds. (Alan R. Liss, New York, 1986), pp. 321-345. and was tested
at this concentration for any deleterious effects on digestion
using electrophoresis. A 7 l volume of the melted sample was
typically pipetted (slowly) using a wide bore pipette tip onto
an 18X18 mm cover glass and rapidly deposited onto a slide.
Timing and quenching of the gel is critical for controlling
elongation. The reaction chamber was then sealed with mineral
oil to avoid evaporation, and the agarose was allowed to gel for
at least 30 minutes at 4 C, prior to diffusion of 50mM MgC12
through an open space. For chromosome I(240kb) and III (345kb),
slides were in a cold desiccator (4 ) prior to casting to hasten
gelling avoiding premature molecular relaxation. For the larger
chromosomes, which relax more slowly, slides were kept at room
temperature. The slide was placed on a temperature controlled
microscope stage at 37 C (except CspI, 30 C). Excessively
stretched molecules present too little fluorochrome per imaging
pixel, so that measured molecular intensities approach
background values. Additionally, the fixation process has to be
gentle enough to permit some coil slippage to reveal cut sites.
Taking these and other considerations into account, our fixation
conditions were optimized to produce molecules spanning
approximately 20t of their curvilinear contour lengths.
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How DNA molecules are entrapped by agarose gelation is
not known. Imaged, stretched molecules show bright round pools
of coil at their ends, evidence of chain relaxation (Figs. 8,
10). The pool sizes range from 1-3 m. Segmental pools are also
observed to form internally, and then disappear, as local
pockets of coil tension equilibrate with each other. As a DNA
molecule relaxes within the train of contiguous gel pores it
spans, the segmental density increases, and segments can even be
seen to spill over into neighboring pore spaces. The detailed
relaxation mechanism is a complex one (de Gennes, et al.,
Scaling Concepts in Polymer Physics, Cornell University Press,
1979). Gaps appear because a molecule experiences an effective
tension since the configurational entropy of the elongated
polymer is lower than that of the relaxed state. On a simple
descriptive level, the process can be compared to watching the
relaxation of a stretched-out thick rubber band encased in a
tight tube, with holes in the sides. Cleavage accelerates
relaxation by creating new ends within a molecule, and possibly
also by causing a mechanical perturbation that releases trapped
fragments from local energy minima.
A high numerical aperture microscope objective can
produce bright, high contrast images of stained DNA molecules,
but with a very shallow depth of focus. Experimentally, for a
long molecules to be in focus, it must lie within a plane
approximately 0.2 m thick. Our method of gel fixation
reproducibly allows visualization of molecules that are within
this 0.;2 micron tolerance as measured optically. This
remarkable degree of optical flatness results from a laminar,
parabolic fluid flow pattern generated between the glass
surfaces, prior to gelation. Furthermore, dissolved agarose and
DNA molecules may potentiate this effect by facilitating laminar
flow, while preventing onset of turbulence (Atkins, 1992).
Finally, gel fixation of large DNA molecules is
convenient enough to be broadly applicable to other systems,
especially when biochemical reactions can be coupled to
visualizable events.
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Restriction Digestion of Single Molecules. Optical
mapping detects restriction enzyme cleavage sites as gaps that
appear in a fixed molecule as fragments relax to a more random
conformation (Figs. 13,15). Since the rates of enzymatic
cleavage by different restriction enzymes are variable (Wells,
et al.,Genetics 127,681, 1981), careful adjustment of the timing
is critical. Cleavage should occur only after molecular
fixation is complete because premature reactions disrupt
attempts to phase fragments. This timing problem was solved by
premixing the agarose-DNA solution with restriction enzyme, at
37 C, and triggering the reaction by diffusing magnesium ions
into the viewing field, without disturbing the gel. Yeast
chromosomal DNA (yeast strain AB972) was resolved by pulsed
electrophoresis (D. C. Schwartz and C.R. Cantor, Cell 37,67
(1984)) using 1.00k Seakem low melting agarose (FMC), 1/2x
TBE(42.5mM Trizma base, 44.5mM boric acid, 1.25mM disodium
EDTA). Cut gel bands were repeatedly equilibrated in TE (10mM
Tris-Cl, imM EDTA, pH8.0). The gel embedded, purified
chromosomes were then equilibrated overnight at 4 C in
magnesium-free restriction buffer containing 0.1 mg/ml
acetylated bovine serum albumin, 1g /3-mercaptoethanol, 0.1t
Triton X-100 (Boehringer Manheim, membrane quality), and 0.2
ug/ml 4', 6-diamino-2 phenylindole dihydrochloride (DAPI) with
slow shaking. Equilibrated samples ranging in volume from 50 to
100 ul were melted at 72 C for 5 minutes, and then cooled to
37 C. Approximately 0.3 - 0.5u1 of enzyme (2 to 14 units/ l)
was spread on a slide. Enzyme reaction temperatures were as
recommended by manufacturers. 0-mercaptoethanol was added to
discourage photolysis M. Yanagida et al. in Applications of
Fluorescence in the Biomedical Sciences, D.L. Taylor et al.,
Eds. (Alan R. Liss, New York, 1986), pp. 321-345. and was tested
at this concentration for any deleterious effects on digestion
using electrophoresis. A 7 l volume of the melted sample was
typically pipetted (slowly) using a wide bore pipette tip onto
an 18X18 mm cover glass and rapidly deposited onto a slide.
Timing and quenching of the gel is critical for controlling
elongation. The reaction chamber was then sealed with mineral
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oil to avoid evaporation, and the agarose was allowed to gel for
at least 30 minutes at 4 C, prior to diffusion of 50mM MgC12
through an open space. For chromosome I(240kb) and III (345kb),
slides were in a cold desiccator (4 ) prior to casting to hasten
gelling avoiding premature molecular relaxation. For the larger
chromosomes, which relax more slowly, slides were kept at room
temperature. The slide was placed on a temperature controlled
microscope stage at 37 C (except CspI, 30 C). Aside from gaps,
cleavage is also signaled by the appearance of bright condensed
pools or "balls" of DNA on the fragment ends at the cut site.
These balls form shortly after cleavage and result from coil
relaxation which is favored at ends (Figs. 13,15). This pooling
of segments is useful in map making because it helps to
differentiate out-of-focus segments, that might appear as gaps,
from actual cuts. Cleavage is scored more reliably by both the
appearance of growing gaps and enlarging bright pools of
segments at the cut site.
Map Construction - Fragment Number Determination.
Large scale restriction maps have been constructed primarily
from electrophoretically derived data. A new set of approaches
has been developed to size and order fragments on samples that
can consist of single DNA molecules, using microscope based
techniques. The first step is to determine the number of
cleavage sites within a molecule. The cut sites within a
molecule tend to appear at irregular times after Mg2+ addition.
All possible cleavage sites do not appear simultaneously;
instead, cuts usually appear within 5 minutes of each other,
under the conditions described here. The extent of digestion
depends on a number of factors including both the fragment
number and size. Digestion results obtained by optical mapping
for a selected set of Not I digested yeast chromosomes are
displayed in Fig. 7. Fortunately, published Not I restriction
enzyme maps are available for all S. cerevisiae chromosomes
(Link, 1991), enabling reliable benchmarking of the optical
mapping methodology.
A typical mounted sample contains approximately 3-5
molecules within a single viewing field and overall, roughly 50-
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95% of them show evidence of one or more cuts by the criteria
described here. The histograms in Fig. 7 show that the overall
number of cut sites exceeding published results is quite low.
The cutting frequency results (Fig. 7B) for chromosome V
digested with Not I show that the number of fully cut molecules
is approximately half that of all singly cut molecules: the
value corresponding to complete digestion is caculated by
assuming that an equal distribution of identically sized
chromosome V and VIII DNA molecules are present in the mounted
sample. The Not I restriction maps for these chromosomes reveal
that chromosome V has 3 cut sites, while VIII has only 2.
Chromosome XI cutting frequency data (Fig. 7C) is different; 25%
of all cut molecules are seen to be fully digested (two cutting
sites). An explanation for the apparently lower frequency is
that this chromosome produces a 30 kb sized Not I fragment that
is more difficult to detect optically than larger fragments.
This result is not surprising considering that tension across a
cut is probably fragment size dependent, so that smaller,
elongated fragments apply less tension. Furthermore, since coil
tension across a cut site is required for its identification,
additional cuts will produce fragments that ultimately relax to
reduce the overall molecular tension and impede the observation
of further cuts. Finally, very large, 1 megabase sized
molecules have been spread, such as chromosome XIII and XVI, and
these data (Fig. 7D) show that roughly half of the molecules are
digested to completion (one cut) in mounts with observable
cutting activity.
The maximum number of cuts determined by histogram
analysis is the bin containing the largest number of cut sites
whose molecules can be properly averaged by intensity and length
measurements for size.
Influence of coil relaxation on detection of cuts.
Aside from cases involving small fragments, incomplete digestion
is seen in all the histograms in Fig. 7. While potential cases
range from photo irradiation artifacts to interactions imposed
by the current design of the microscope chamber, partial
digestion observed here is attributable mostly to incomplete
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coil relaxation at a given cut site, due to relaxation modes
that fail to produce a gap or distinct ball. A variety of
different relaxation modes are observed in actual practice, some
of which are sketched in Fig. 8. Relaxation modes can both
facilitate (8-D) and hinder cut detection (8-H). Application of
electric or flow fields might be used to trigger relaxation at
such sites and permit their detection. Parallel electrophoresis
experiments show essentially complete digestion under similar
experimental conditions (Hernandez).
Interestingly, the data for chromosome I show almost
complete digestion (95t; see Fig. 7A). Images of chromosome I
under digestion (Fig. 13A) reveal that after the expected single
cut is observed, only the cut site ends relax and bright pools
of segments accumulate at the ends (20 molecules), as
interpreted in Fig. 8B, BC and SD, while the remaining ends
appear to be fixed in place. Bright pools of relaxed coil
segments accumulate at the ends of gel-fixed DNA molecules, as
noted above.
Conceivably, the ends of chromosome I embedded in
agarose are behaving as a sort of molecular rivet (Fig. 9),
reacting to the tension developed between it and the intervening
molecular segments to provide ideal mechanical conditions for
cut detection. It seems likely that short-range interactions
will predominate so that the amount of relaxed coil present at
the ends of elongated molecules will not vary much with
molecular mass above some threshold in size. Consequently, a
relatively short molecule, such as chromosome I, will contain a
greater proportion of relaxed coil segments at its end than
longer ones, such as chromosomes XIII and XVI.
Fragment Sizing By Relative Intensity. The second
step is to size the resulting restriction fragments. For this
purpose two complementary approaches can be used, one based on
relative fragment fluorescence intensity and the second on
apparent relative length measurements. However, neither
approach provides absolute values, but each can be standardized
readily. Fortunately, the gel fixation technique described
above produces a natural substrate for intensity measurements
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since an entire molecule can be brought into focus. Gel
fixation is able to flatten molecules spanning as much as 250
m. Segments of molecules that are out of focus cannot be used
for intensity measurements because their intensities are not
proportional to mass in any simple way. A relevant observation
here is that when an elongated molecules substantially relaxes,
most of its mass moves out of focus, as expected, since the
hydrodynamic diameter of a fully relaxed 700 kb DNA molecule in
fluid is 8 m while the depth of focus used for imaging
molecules under the microscope is approximately 0.2 m.
The absolute fluorescence intensity of a DNA fragment
in the microscope is determined by many variables, such as the
camera gain control and lamp brightness, and therefore is not a
desirable quantity to measure. By calculating the relative
intensity of two fragments (from the same parental molecule),
one of the fragments can serve as an internal intensity
reference for the other. Relative intensities are converted to
kb by multiplying by the know or independently determined
chromosome size. Length and relative fluorescence intensity
were calculated to 16-bit precision using a modified version of
NIH Image for Macintosh by Wayne Rasband, available upon request
from the authors (e-mail huff @ mcclb0.med.nyu.edu). Further
details are available (manuscript in preparation). Briefly, the
original unprocessed image was displayed in an enlarged format
and an overlay image was prepared by manually tracing the DNA.
The length map was made directly from this overlay. For
intensity calculations, the 13-bit raw data image was smoothed
and the overlay image was dilated five times to cover all
foreground pixels. For each pixel marked on the overlay, a
synthetic background value was calculated as the weighted
average of surrounding pixels, with a weight that decreased with
distance, but was zero for all marked pixels. These values are
intended to approximate those which would have been measured had
the DNA been absent. The intensity of a particular DNA fragment
was the sum of all pixels of the fragment minus the matching
background pixels. The area of the fragment was the original
overlay dilated twice. This process was repeated for each frame
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of raw data which had an overlay image, excluding those with
poor focus. Intensity results were averaged for five images
following a cut, and the relative sizes of the two fragments
were calculated as x/(x+y) and y/(x+y). If fragment y later
cuts into u and v, then (y/(x+y))(u/(u+v)) is used for the size
of u. The resulting numbers constitute a single sample for the
purposes of subsequent analysis. The optical contour
maximization technique can be used to size samples containing a
small number of molecules (Guo, Nature 359,783, 1992). Fig. 10A
shows intensity values for a series of yeast chromosome Not I
restriction fragments measured optically and plotted against
published values derived from electrophoresis based measurements
(Link, Genetics, 127, 681, 1991). Points close to the diagonal
line are in good agreement. Disregarding the chromosome V and
VIII results, which were based on low precision (8-bit)
intensity data, and disregarding the two short fragments less
than 60kb, the pooled standard deviation is 36kb (Fig. 5A inset)
and the average of the coefficients of variation is 16t,
comparable to routine pulsed electrophoresis size
determinations. The correlation with published results is
excellent: the average of the relative errors is 5k whereas the
published errors average 4t (Link, Genetics, 127, 681, 1991).
The samples were averaged and the 90t confidence interval on the
mean was calculated using the t distribution with n-i d.f. and
the sample standard deviation. This calculation is valid if the
data represent random samples from a normal distribution. There
is a 90t chance that the population mean falls within the
confidence interval. For chromosome I, the reported confidence
interval was found by taking the lower bound from the short
fragments and the upper bound from the long fragments. The 90t
confidence interval for the population standard deviation (Fig.
10 inset graphs) was calculated using the sample standard
deviation, the number of samples, and the chi-square
distribution with n-1 d.f. The midpoint of this interval was
used to estimate the population standard deviation. The
coefficient of variation (CV) is the estimated population
standard deviation divided by the sample mean. The pooled
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standard deviation is the square root of the average of the
variances. The relative error is the differences between our
value and the reported value divided by the reported value. Due
in part to the intensity normalization procedure, the precision
becomes lower for very small fragments, and size agreement is
poor for the 30 and 55 kb measurements. Fluorescence intensity
measurements size these fragments at almost twice the
established values as described below. Changes in the algorithm
for correcting the backgrounds of these measurements and the
data collection process should improve the precision
significantly.
One test of the validity of relative fluorescence
intensity measurements is to monitor the constancy of fragment
intensities over a usable range of molecular relaxation
conditions. This requirement is most critically tested when
restriction fragments differ greatly in size. Fig. 11 shows the
results of absolute intensities versus molecular length
measurements for three typical sizes. These results show that
intensities remain relatively constant over a wide size range
despite a 3-4 fold change in measured molecular length. This
beneficial effect is attributed in part to the mild fixation
conditions, so that Brownian motion can dither the elongated
coil along the z-axis; this motion is clearly observed on the
live video monitor as digestion proceeds. By averaging frames
over a 1 second interval most of the DNA is observed as it moves
through the focal plane and within the gel pores.
Fragment Sizing by Relative Apparent Lengths. The
physical basis of apparent length measurement is simple: each
gel-embedded restriction fragment is assumed to have equal coil
density, on the average. That is, each fragment has the same
change to be stretched more or less, so a length average created
over a number of mounts provides a good measure of relative
size. Again, relative apparent lengths are converted to kb by
multiplying by the chromosome size. Length and relative
fluorescence intensity were calculated to 16-bit precision using
a modified version of NIH Image for Macintosh by Wayne Rasband,
available upon request from the authors (e-mail huff @
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mcclb0.med.nyu.edu). Further details are available (manuscript
in preparation). Briefly, the original unprocessed image was
displayed in an enlarged format and an overlay image was
prepared by manually tracing the DNA. The lengr.h map was made
directly from this overlay. For intensity calculations, the 13-
bit raw data image was smoothed and the overlay image was
dilated five times to cover all foreground pixels. For each
pixel marked on the overlay, a synthetic background value was
calculated as the weighted average of surrounding pixels, with a
weight that decreased with distance, but was zero for all marked
pixels. These values are intended to approximate those which
would have been measured had the DNA been absent. The intensity
of a particular DNA fragment was the sum of all pixels of the
fragment minus the matching background pixels. The area of the
fragment was the original overlay dilated twice. This process
was repeated for each frame of raw data which had an overlay
image, excluding those with poor focus. Intensity results were
averaged for five images following a cut, and the relative sizes
of the two fragments were calculated as x/(x+y) and y/(x+y). If
fragment y later cuts into u and v, then (y/(x+y))(u/(u+v)) is
used for the size of u. The resulting numbers constitute a
single sample for the purposes of subsequent analysis. Then,
the apparent lengths of restriction fragments are converted,
obtaining good accuracy from as few as 4 molecules. The samples
were averaged and the 90% confidence interval on the mean was
calculated using the t distribution with n-1 d.f. and the sample
standard deviation. This calculation is valid if the data
represent random samples from a normal distribution. There is a
90%- chance that the population mean falls within the confidence
interval. For chromosome I, the reported confidence interval was
found by taking the lower bound from the short fragments and the
upper bound from the long fragments. The 90% confidence
interval for the population standard deviation (Fig. 10 inset
graphs) was calculated using the sample standard deviation, the
number of samples, and the chi-square distribution with n-1 d.f.
The midpoint of this interval was used to estimate the
population standard deviation. The coefficient of variation
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(CV) is the estimated population standard deviation divided by
the sample mean. The pooled standard deviation is the square
root of the average of the variances. The relative error is the
differences between our value and the reported value divided by
the reported value. Relative determinations of apparent length
were verified against the same set of restriction fragments as
in the fluorescence intensity measurements, and these results
(Fig. lOB) show a similar average relative error of 16t (exclud-
ing the 30 and 90kb fragments). The pooled standard deviation
was 47kb (Fig lOB inset), the average of the coefficients of
variation was 29g.
Apparent molecular length measurements are more robust
than intensity measurements, but are less precise, and
consequently require additional measurements to achieve an
equivalent degree of accuracy. But good length measurements can
be obtained from slightly out-of-focus fragments, whereas
blurry, out of focus images will confound intensity based
measurements. Size determination of small fragments by length
were better than intensity. The 30kb fragment was sized at 44kb
by length vs. 70kb by intensity, and the 55kb fragment was sized
at 49kb vs. 88kb. Given the limited sample number inherent to
optical mapping, having two sizing methods for cross-checking
results is extremely important for successful map making.
Map Construction Based on Length and Intensity
Measurements. Fig. 12 illustrates three types of ordered
restriction maps produced by optical mapping compared with
(Link, Genetics 127, 681, 1991). The bars shown correspond to
sizing analysis results of the Not I restriction fragment as
plotted in Fig. 10. Fig. 13 shows selected processed
fluorescence micrographs of different yeast chromosomal DNA
molecules digested with Not I. Yeast chromosomal DNA (yeast
strain AB972) was resolved by pulsed electrophoresis (D. C.
Schwartz and C.R. Cantor, Cell 37:67 (1984)) using 1.00t Seakem
low melting agarose (FMC), 1/2x TBE(42.5mM Trizma base, 44.5mM
boric acid, 1.25mM disodium EDTA). Cut gel bands were
repeatedly equilibrated in TE (10mM Tris-Cl, imM EDTA, pH8.0).
The gel embedded, purified chromosomes were then equilibrated
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overnight at 4 C in magnesium-free restriction buffer containing
0.1 mg/ml acetylated bovine serum albumin, 1t fl-mercaptoethanol,
0.1%- Triton X-100 (Boehringer Manheim, membrane quality), and
0.2 ug/ml 41, 6-diamino-2 phenylindole dihydrochloride (DAPI)
with slow shaking. Equilibrated samples ranging in volume from
50 to 100 ul were melted at 72 C for 5 minutes, and then cooled
to 37 C. Approximately 0.3 - 0.5u1 of enzyme (2 to 14 units/ l)
was spread on a slide. Enzyme reaction temperatures were as
recommended by manufacturers. fl-mercaptoethanol was added to
discourage photolysis (M. Yanagida et al. in Applications of
Fluorescence in the Biomedical Sciences, D.L. Taylor et al.,
Eds. (Alan R. Liss, New York, 1986), pp. 321-345.) and was
tested at this concentration for any deleterious effects on
digestion using electrophoresis. A 7 1 volume of the melted
sample was typically pipetted (slowly) using a wide bore pipette
tip onto an 18X18 mm cover glass and rapidly deposited onto a
slide. Timing and quenching of the gel is critical for control-
ling elongation. The reaction chamber was then sealed with
mineral oil to avoid evaporation, and the agarose was allowed to
gel for at least 30 minutes at 4 C, prior to diffusion of 50mM
MgC12 through an open space. For chromosome I(240kb) and III
(345kb), slides were in a cold desiccator (4 ) prior to casting
to hasten gelling avoiding premature molecular relaxation. For
the larger chromosomes, which relax more slowly, slides were
kept at room temperature. The slide was placed on a temperature
controlled microscope stage at 37 C (except CspI, 30 C). These
images clearly show progressive digestion by the appearance of
growing gaps in the fixed molecules. From such data fragment,
order was determined from inspection of time-lapse images
obtained every 20 seconds. DNA molecules were imaged using a
Zeiss Axioplan or Axiovert 135 microscope equipped for epi-
fluorescence (487901 filter pack for UV excitation and Blue
emission) and a 100X or 63X Plan-Neofluar objective (Zeiss)
coupled to Hammamatsu C2400 SIT cameras. Care was taken to
adjust the camera controls to avoid saturating the digitizer at
either end of the intensity range. Every 20 seconds, 32 video
frames were digitized to 8 bits and integrated to give 13 bit
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precision by a Macintosh based Biovision image processor or a
Pixel pipeline digitizer (Perceptics Corp.). A computer
controlled shutter was used to limit illumination to 1.5 seconds
per image giving a total of about 135 to 255 seconds for typical
experiments. Neutral density filters were used to keep the
illumination intensity below 100 W measured at the objective.
Control experiments showed no damage to DNA molecules under
these conditions. Digitized images were recorded directly to
disk and archived on tape. Since observed molecules tend to
move and can sometimes be confused with other molecules,
inspection of a "cutting sequence" or "cutting movie" simplifies
deconvolution of molecule-molecule interactions. Agreement is
excellent between the optical (length or intensity) and the
electrophoresis based maps. The third type of restriction maps
("Com", Fig. 7) results from combining length and intensity
derived data: data from small restriction fragments (<60kb)
were sized by length, while intensity measurements provide the
balance of fragment sizes needed to complete the maps.
Fig. 14 shows the ordered restriction maps created
from Rsr II digestion of chromosome III and XI and Asc I
digestion of chromosome XI by optical mapping, while Fig. 15
shows the corresponding fluorescence micrographs of typical
digests. Relative apparent length results, using the pooled
population standard deviation of 47kb to calculate confidence
intervals. Chromosome, enzyme, mean +/- 90% confidence kb
(number of samples). Ch. III Rsr II 264 +/- 27(8), 86 +/-
27(8). Ch. XI Asc I 42 +/- 55(2), 195 +/- 55(2), 242 +/- 55(2).
Ch. XI Rsr II 67 +/- 45(3), 127 +/- 45(3), 221 +/- 45(3), 260
+/- 45(3). Relative fluorescence intensity results, using the
pooled population standard deviation of 36kb to calculate
confidence intervals. Ch. III Rsr II 256 +/- 21(8). Ch. XI Asc
I 80 +/- 42(2), 177 +/- 42(2), 181 +/- 42(2), 237 +/- 42(2).
Ch. XI Rsr II 84 +/- 34(3), 125 +/- 34(3), 226 +/- 34(3), 240
+/- 34(3). There are no published maps available for
independent verification of these results. These maps are
constructed by first determining the maximum number of cleavage
sites from cutting frequency data (similar to Fig. 7).
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Fragments from fully cut molecules are then siaed'by length and
intensity and sorted into bins for averaging. Relative
fluorescence intensity measurements are used to sort length
measured fragments. Obviously, adjacent fragments must go into
adjacent bins for averaging. Distinctive patterns in a digest,
such as a very large fragment lying next to a very small one,
facilitate accurate sorting. Data from partial digests was also
used to confirm the maps. Data from partial digests was used to
confirm the map constructed from fully cut molecu].es by
calculating the expected partial fragment lengths and comparing
these to the observed data.
A new set of analytical approaches to physical mapping
of very long molecules, such as DNA molecules, is thus provided
according to the present invention, that is simple and
intrinsically very rapid. A nearly real time mapping procedure
for chromosomes of yeast has been implemented, but this is far
from the ultimate capability of the methodology. Since most
traditional tools of genomic analysis are bypassed, including
cloning, electrophoresis, Southern analysis and PCR, additional
speed increases in optical mapping are not predicated on
advances in robotics or automation (Chumakov, Mture 359:380,
1992). Simple engineering advances in chamber design, sample
handling, image analysis and informatics should make available a
high throughput methodology capable of rapidly mapping entire
genemes and, more importantly, extending knowledge of sequence
information to populations of individuals rather than prototypes
=of each organism (Cavalli-Sforza, Am. J. Hum. Genet 46:649,
1990).
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Reference to known method steps, conventional methods
steps, known methods or conventional methods is not in any way
an admission that any aspect, description or embodiment of the
present invention is disclosed, taught or suggested in the
relevant art.
The foregoing description of the specific embodiments
will so fully reveal the general nature of the invention that
others can, by applying knowledge within the skill of the art
(including the contents of the references cited herein), readily
modify and/or adapt for various applications such specific
embodiments, without undue experimentation, without departing
from the general concept of the present invention. Therefore,
such adaptations and modifications are intended to be within the
meaning and range of equivalents of the disclosed embodiments,
based on the teaching and guidance presented herein. It is to
be understood that the phraseology or terminology herein is for
the purpose of description and not of limitation, such that the
terminology or phraseology of the present specification is to be
interpreted by the skilled artisan in light of the teachings and
guidance presented herein, in combination with the knowledge of
one of ordinary skill in the art.
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