Note: Descriptions are shown in the official language in which they were submitted.
- 208g~2~ ~
DNA FRAGMENT SIZING AND SORTING
BY LASER-INDUCED FLUORESCENCE
BACKGROUND OF THE INVENTION
The human genome is comprised of some three billion
nucleotides forming the 22 pairs of chromosomes plus 2
autosomes, each with continuous DNA pieces of 50-500
; million nucleotides. The organization and sequence of DNA
' forming the human genome contains unique information about
; the source that provides the DNA. One method for accessing
this information is to fragment the DNA at sites with known
characteristics and then to analyze the distribution of
fragment sizes, i.e., the number of nucleotides in each
fragment between each of the sites. Polymorphisms in the
genome structure lead to substantial variation in the
fragment sizes obtained from fragmentation of DNA pieces
and allow one to differentiate one person from another or
to form a basis for assessing a person's susceptibility to
genetic diseases. Analysis of these polymorphisms is often
referred to ~s DNA f ngerprinting.
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2089423
,
DNA fingerprinting is an important medical diagnostic
tool, with additional applications to forensic
identification, medical genetics, monitoring the effects of
environmental mutagens, and basic molecular biology
research. One form of DNA fingerprinting involves
"restriction fragment length polymorphism" (RFLP) where
restriction enzymes are used to cut a DNA piece from a
specific source into shorter pieces, or fragments, of DNA.
RFLP provides a unique pattern of DNA fragments containing
a unique DNA sequence ordered by fragment size (the DNA
fingerprint) when a DNA specimen is digested with
restriction enzymes. There are many known restriction
enzymes and each recognizes a specific DNA sequence of four
to twelve base pairs at which it cuts the DNA , resulting
in smaller fragments of DNA.
Once the DNA piece has been cut into many fragments,
electrophoresis is conventionally used to separate the
fragments by size. An electric field is placed across a
gel containing the fragments causing the smaller fragments
to move faster than the larger ones. Gel electrophoresis
is a well known technique and has been used to produce band
patterns of DNA fragments that form a fingerprint to
identify the individual source of the DNA piece under
analysis. The band patterns of specific DNA sequences are
conventionally visualized by binding radioactive DNA probes
to the separated DNA fragments and exposing suitable film
to the radioactive labeled fragments. See, e.g., J.I.
Thornton, "DNA Profiling," C&EN, pp. 18-30 (November 20,
1989); K. Heine, "DNA on Trial," Outlook 26:4, pp. 8-14
(1989)- In one variation, the fragment ends are tagged
with a fluorescent dye so that the fragment migration time
along a known path length in an electrophoretic gel can be
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determined by automated fluorescence detection. See, e.g.,
- A.V. Carrano, "A High-Resolution, Fluorescence-Based,
Semiautomated Method for DNA Fingerprinting," 4 Genomics,
pp. 129-136 (1989).
There are, however, several limitations on the use of
yel electrophoresis, particularly where large fragment
sizes and radioactive labeling are involved. In both
instances, the electrophoretic separation process takes
considerable time to provide resolution for large size
fragments. The development of images from radioactive
probes is an additional time consuming step and has health
hazards and environmental concerns associated with
radioactive materials. Additionally, the distribution of
fragment sizes is logarithmic so that the separation, i.e.,
resolution, between large fragments is less than for small
fragments. Electrophoresis also requires relatively large
amounts of DNA to obtain a recognizable pattern.
It is desirable to provide a DNA fragment size analysis
- technique that uses only small quantities of DNA (maybe
only a single strand), provides size information within a
short time, and has a high resolution between fragment
sizes. These and other problems of the prior art are
addressed by the present invention wherein flow
cytometry-based techniques are used to obtain a
distribution of DNA fragment sizes from a DNA piece.
Accordingly, it is an object of the present invention
to provide rapid determination of DNA fragment sizes.
It is another object of the present invention to obtain
a high resolution of DNA fragments, particularly long
fragments.
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20489423
One other object of the present invention is to require
only a small DNA sample to provide accurate DNA fragment
size fingerprints.
Yet another object of the present invention is to
enable fragment length detection without the use of
radioactive labels.
A further object of the present invention is to use
fluorescent intensities to determine the length of DNA
fragments.
Still another object of the present invention is to use
~ the sorting capabilities associated with flow cytometry to
-~ sort the fragments by size, i.e., length, for further
- study.
. Additional objects, advantages and novel features of
- 15 the invention will be set forth in part in the description
which follows, and in part will become apparent to those
skilled in the art upon examination of the following or may
' be learned by practice of the invention. The objects and
advantages of the invention may be realized and attained by
means of the instr~mentalities and combinations
particularly pointed out in the appended claims.
SUMMARY OF THE INVENTION
To achieve the foregoing and other objects, and in
accordance with the purposes of the present invention, as
embodied and broadly described herein, the method of this
invention may comprise the use of an induced fluorescence
to quantitate the length of DNA fragments. A piece of DNA
is fragmented at preselected sites to produce a plurality
of DNA fragments. All of the DNA fragments are treated
; 30 with a dye effective to stoichiometrically stain the
nucleotides along the DNA fragments. The stained DNA
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~089423
fragments are then fluorescently examined to generate an
output functionally related to the number of nucleotides in
each one of the DNA fragments. In one output, the
intensity of the fluorescence emissions from each fragment
is directly proportional to the fragment length.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, DNA
polymorphisms are characterized, i.e., "fingerprinted,"
using flow cytometry-based techniques to provide a rapid
analysis of DNA fragment sizes obtained by fragmenting a
- selected DNA piece with one or more enzymes selected to
cleave DNA at known sequence sites. One exemplary
procedure for sizing is the following:
1. A DNA piece from a selected source is fragmented
by enzyme digestion to provide a solution of DNA
fragments. The nucleotides comprising the DNA piece may be
stained, i.e., labeled, with an appropriate fluorescent dye
either before or after the DNA piece is fragmented.
2. The stained DNA fragments are passed through a
detection apparatus at a concentration and rate effective
to provide only one fragment in the fluorescence excitation
volume at any one time.
3. Each stained DNA fragment is excited, e.g., with
laser irradiation, in the excitation volume and the
resulting fluoresence intensity is measured, wherein the
intensity of the induced fluorescence is a measure of the
amount of stain on the fragment and concomitant fragment
` length.
4. The number of fragments at each different
intensity provides an analysis of the number of fragments
`~ of each length produced from the DNA piece by the selected
enzyme or enzymes.
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6 - 2~89423
A DNA piece may be first selected from any suitable
source, e.g., blood, tissue samples, semen, laboratory
research specimens, etc. The DNA piece is then fragmented
using an enzyme chosen for a particular application of the
analysis. One particularly useful type of enzyme is a
restriction endonuclease that recognizes specific sites,
i.e., specific nucleotide sequences, and cleaves the DNA
piece within the identified sequence. For example, the
enzyme Eco RI cuts at the double piece recognition site
--GAATTC---
--CTTAAG---
Hundreds of different restriction enzymes and their
respective cleavage sites are known. It will be
appreciated that identical DNA pieces from a single source
might be digested with different enzymes to yield a family
of fingerprints. Alternatively, a DNA piece may be
digested with multiple enzymes to further particularize the
fragment size distribution analysis.
Fragmentation, i.e., digestion, of a DNA piece with a
selected enzyme is a well-known process, where the optimum
digestion conditions are specified by the enzyme
manufacturer. A generic restriction enzyme process for use
with 0.2-1 ~g of DNA is given by J. Sambrook et al.,
Molecular Cloning, pp. 5.28-5.33, Cold Spring Harbor
Laboratory Press (1989):
1. Place the DNA solution in a sterile microfuge tube
and mix with sufficient water to give a volume of 18
~L.
2. Add 2 ~L of an appropriate restriction enzyme
digestion buffer and mix by tapping the tube. An exemplary
~ buffer may be formed as follows:
; 200 mM potassium glutamate
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2089423
50 mM Tris-acetate tpH 7.5)
20 mM magnesium acetate
100 ~g/mL bovine serum albumin (Fraction V; Sigma)
1 mM ~-mercaptoethanol.
3. Add 1-2 units of restriction enzyme and mix by
tapping the tube, where 1 unit of enzyme is defined as the
amount required to digest 1 ~g of DNA to completion in
1 hour in the recommended buffer and at the recommended
temperature in a 20-~L reaction.
4. Incubate the mixture at the appropriate
temperature for the required period of time.
5. Stop the reaction by adding 0.5 M EDTA (pH 8.0) to
a final concentration of 10 mM.
DNA can be fragmented by a variety of other techniques
in addition to restriction enzyme digest:
1. DNase hypersensitivity sites (DNase
footprinting). Chromatin digestion by DNase will produce
fragments of various lengths due to differences in proteins
; that bind to the DNA and prevent cutting of the DNA by a
DNase at sites where protein is bound (D.J. Galas et al.,
Nucleic Acids Res., 5:3157 (1987)).
2. RNase cleavage of single-base pair mismatches. A
fluorescent RNA probe is synthesized complementary to a
normal, or wild type, DNA sequence of interest. This
complementary probe is then annealed to the target DNA that
is to be analyzed. To determine if a single nucleotide
mismatch exists between the fluorescent probe strand and
the target DNA strand, the RNA:DNA hybrid is treated with
RNase A. RNase A specifically cleaves single stranded
regions of RNA, thus cleaving the single base pair mismatch
region in the fluorescent RNA strand of the RNA:DNA hybrid.
(See R.M. Myers et al., Science 230:1242 (1985) and E.F.
Winter et al., Proc. Natl. Acad. Sci. 82:7525 (1985)).
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2089~23
3. RecA-assisted restriction endonuclease cleavage.
Short oligonucleotides coated with RecA protein are
annealed to the complementary target DNA sequence. The
DNA:oligonucleotide hybrid is treated with Eco RI methylase
enzyme. Eco RI sites that are not protected by the
oligonucleotide are methylated while oligonucleotide
protected Eco RI sites remain unaffected. Eco RI
restriction endonu~lease will cleave only at protected
sites (i.e., unmethylated). This method has been used to
generate fragments>500,000 base pairs. (L.J. Ferrin,
Science, 254:1494 (1991).
4. DNA fragmentation can also be accomplished by
techniques other than enzyme digestion. For example,
ultrasonic excitation at different fre~uencies might be
used to produce a family of size distributions. Various
chemicals also react with the nucleotides and may be used
to fragment DNA pieces.
The DNA fragments must be stained with a fluorescent
dye for flow cytometric analysis. A fluorescent dye is
selected to bind stoichiometrically to the DNA fragments.
; The complex may be formed in different ways, i.e., single
' stranded DNA, double stranded DNA, specific base pairs,
etc. Well known dyes include ethidium bromide, acridine
orange, propidium iodide, DAPI, Hoechst, chromomycin,
mithramycin, 9 amino acridine, ethidium bromide heterodyne,
- asymmetric cyanine dyes, or combinations of these dyes for
energy transfer. The selected dye or dyes bind to the
oligonucleotides stoichiometrically along the DNA sequence,
i.e., the binding sites along the fragments are such that
the total number of dye molecules along any length of DNA
is proportional to the number of base pairs (bp's) forming
the DNA. For example, under the staining protocol set
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2~8~423
forth below, the number of ethidium bromide molecules bound
to a DNA fragment is stoichiometric and can be as high as
one-half the number of bp's. See, e.g., C.R. Cantor et
al., "Binding of Smaller Molecules to Nucleic Acids," in
~iophysical Chemistry~ Part III: The Behavior of Bioloqical
Macromolecules, p. 1251, W.H. Freeman and Company, 1980.
One exemplary procedure for staining with ethidium
~romide is:
Add DNA sample to a solution containing 1-5 ~g of
ethidium bromide per mL of solution and TE 8.0 buffer.
A suitable buffer is available from GIBCO and is 10 mM
Tris-HCL and 1 mM EDTA, pH 8Ø The reaction is
complete in 5-10 minutes at room temperature.
The stained DNA fragments are now analyzed using
sensitive fluorescence detection techniques to determine
the fluorescence intensity from each fragment passing
through a detection region and having a high resolution to
distinguish adjacent fragment sizes. Theoretically, while
only a single DNA piece is needed to obtain the desired
distribution analysis, a typical solution will be formed
from many DNA pieces and a relative DNA fragment size
distribution is obtained. The DNA fragments will typically
range in size from 100 bp to 500,000 bp.
~ It is well-known how to form a sequential flow stream
- 25 of particles for use in a flow cytometer or similar
sensitive fluorescence detection apparatus. See, e.g.,
U.S. Patent 3,710,933, issued January 16, 1973, to Fulwyler
et al. and Flow Cytometry and Sortinq, 2nd Ed., ed. M.R.
Melamed et al., Wiley-Liss, New York, 1990, incorporated
herein by reference. A dilute solution of the DNA
fragments is formed to a low concentration effective to
provide the fragments spaced apart in the flow stream so
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2089423
'' 10
that only a single fragment is present in the excitation
volume. The solution of DNA fragments is then injected
within a laminar sheath flow stream for passage through the
detection chamber for laser excitation of one fragment at a
time. The flGw rates of the sample and the sheath are
adjusted to maintain separation between particles and to
provide the optimum time for each particle in the
excitation source. An optimum time is determined from a
consideration of sizing rate, detection sensitivity, and
photostability of the dye tags. A suitable excitation
source is selected to initiate fluoresce in the dye used to
stain the DNA fragments. For example, an argon laser at
488 nm is effective to cause ethidium bromide to fluoresce
in a band around 600 nm.
The sensitivity of conventional flow cytometry system
is improved by providing a small excitation volume, e.g.,
- 10-20 ~m diameter and 100 ~m length, with a tightly
focused laser beam. See, e.g., J.H. Hahn et al.,
"Laser-Induced Fluorescence Detection of Rhodamine-6G at
6x~0 15M," Appl. Spectrosc. 45:743 (1991), describing a
probe volume of 11 pL, incorporated herein by reference.
The small probe volume greatly reduces the amount of
background emission, i.e., noise, in the output signal.
The laser excitation may be a pulsed laser with a pulse
of e.g., about 70ps full width, with time gating to
differentiate between dye emission photons (delayed) and
Raman scattering photons (prompt). See, e.g., E.B. Shera
et al., "Detection of Single Fluorescent Molecules," Chem.
Phys. Lett. 174:553 (November 1990), The prompt scattered
photons occur within the laser pulse time while the dye
emissions decay with a several nanosecond lifetime so that
a delayed window is effective for discrimination of
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2089423
11
fluorescence photons from Raman scatter photons.
Alternatively, the laser may be a cw laser. See, e.g.,
S.A. Soper, "Single-Molecule Detec ion of Rhodamine-6G in
Ethanolic Solutions Using Continuous Wave Laser
Excitation," Anal. Chem. 63:432 (1991). The number of
emitted photons can be increased by increasing the DNA
fragment transit time through the laser beam and by
selecting a dye and solvent with high photostability for
the dye. The number of detected photons (photoelectrons)
is also increased by increasing the sensitivity of the
detection apparatus. Furthermore, the present invention
-~ involves DNA fragments rather than single molecules so thatthe longer fragment length so that a larger output
fluorescence intensity is obtained.
,~ 15 It will also be appreciated that the solution may
! ~ contain some dye that was not bound to the DNA fragments.
~his dye will be excited along with bound dye and the
system must discriminate between the fluorescence from the
: unbound and the bound dye. In one embodiment, a pulsed
laser and gated detection technique may be used to provide
this discrimination. For example, the excited state
lifetimes for the unbound and bound ethidium bromide are 2
ns and 23 ns, respectively. Thus, the detection system can
be gated to detect only the fluorescence from the bound
ethidium bromide and, hence, provide an output signal
~! ~ functionally related to the length of the DNA fragment.
` Alternatively, a dye might be selected that provides
:~~ different fluorescence or absorption wavelengths in the
bound and unbound states. For example, a series of
asymmetric cyanine dyes are reported by I.D. Johnson et
~ al., "Asymmetric Cyanine Dyes for Fluorescent Staining and
; Quantification of Nucleic Acids," Fluorescence
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2089423
12
~ Spectroscopy, Abstract 1806, FASEB J. 6:A314, No.
- (January 1992).
Polarized fluorescence emission also provides a means
of discriminating bound from unbound dye molecules.
F:Luorescence polarization of unbound DNA dyes is <0.05,
whereas the fluorescence polarization of DNA bound
f:Luorochromes can be between 0.20 and 0.30. See, e.g.,
L.S. Cram et al., "Fluorescence Polarization and Pulse
. Width Analysis of Chromosomes by a Flow System," J.
Histochem. Cytochem. 27:445, No. 1 (1979); T.M. Jovin,
"Fluorescence Polarization and Energy Transfer: Theory and
Application," Flow Cytometry and Sorting, Ed. M.R. Melamed
et al., pp. 156, John Wiley & Sons (1979). Discrimination
is accomplished by using a polarized excitation source and
detecting the emissions through a polarization filter
placed in front of a fluorescence detector. The
polarization filter is aligned with the polarization
direction parallel to the polarization direction of the
excitation source.
For continuous cw laser excitation, an energy
transfer-type scheme may be used to distinguish bound and
unbound dye molecules. If a second dye is also bound to
the DNA, the bound first dye molecules used for fragment
sizing will be in a close proximity to the second dye
molecules so that excitation of the second dye molecules
will result in energy transfer from the second dye
molecules to the first dye molecules. The unbound first
and second dye molecules in the surrounding fluid will not
be in proximity effective for energy transfer. Thus, only
the bound first dye molecules will fluoresce for fragment
length determination when the second dye molecules are
excited.
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2089423
13
By way of example, the DNA specific dyes Hoechst and
chromomycin can transfer energy. When Hoechst dye
`- molecules are excited they can transfer energy to
chromomycin molecules within a transfer radius of a few
angstroms. This energy transfer pair is used in
fluorescent analysis of chromosomes by flow cytometry.
See, e.g., R.G. Langlois et al., "Cytochemical Studies of
Metaphase Chromosomes by Flow Cytometry," Chromosoma 77:229
(1980). ~xcitation by energy transfer may also be done by
exciting the nucleotides at around 260 nm with subsequent
.~ energy transfer to the bound dye molecules. See, e.g.,
J.B. LePecq et al., "A Fluorescent Complex between Ethidium
Bromide and Nucleic Acids," J. Mol. Biol. 27:87 (1967).
The flow cytometry-type apparatus also provides a high
resolution to distinguish between adjacent fragment
lengths. Indeed, the resolution is generally limited by
shot noise in the photons arising from the fluorescense
emissions and the percent resolution increases as the
number of base pairs (bp's) forming the DNA fragments
increases.
; The percent resolution (R) is determined by the length
i of the frag~ent (L), the fraction of the fragment tagged
(f), the number of photoelectrons collected per tag (b) (b
is typically about 30), and the number of times a fragment
is sized (N). The mean intensity is given by ~. The
percent resolution R at a standard deviation of 3O is
given by
`i R(%) = 3*100*Nl/2*(L*f*b*)l/2/(L*f*b*), where
i ~ = L*f*b; O 5 ~1/2.
~: 30 For example, consider the case of a fra~ment 1000 bases
long. L=1000, f=0.5 (for e.g., ethidium bromide), b=30.
For N=l, ~=15000, o=122.474~ and R=2.45%. The
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resolution improves as Nl/2. For N=1000, o=3.87 and
R=0.0775%. Sizing 10, 100, or even 10000 identical
,` fragments is not a problem. There are many more fragments
than 1~000 in a typical electrophoresis band. Thus, it can
be seen that the resolution can be much better than 1% on a
100,000 bp fragment, whereas a resolution of only 10-20%
would be expected for separation of fragments in the
100,000 bp range by gel electrophoresis and the resolution
degrades further as fragment length increases.
DNA fingerprinting according to the present invention
can also be done very rapidly. A typical DNA fingerprint
by electrophoresis has about 50 bands. At 1000 fragments
per band, 50 bands would require only about 8.3 minutes to
develop a fingerprint at a fragment analysis rate of 100
fragments/second. If only a single band is required for
the desired resolution, the analysis time would be only
about 1 second. If the desired resolution requires 100
fragments per band, then the 50 band analysis would take
only about 50 seconds.
In an adaptation of the present invention,
hybridization probes can be bound to the DNA restriction
;; fragments and associated with fragment length sizes.
Hybridization probes are conventionally formed containing a
`'i probe dye and hybridized to DNA fragments formed by base
pair matching from the DNA piece being investigated.
Excitation of the hybridized DNA fragments could then be
designed to excite both the size-measuring dye and the
probe dye so that correlation of the fluorescent outputs
would associate the probe with various fragment lengths.
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,~ 30 In situ probe hybridization to DNA is discussed in Methods
in Cell Biology, Vol._ 33, Z. Darzynkiewicz et al. Ed.,
Academic Press, Inc. ~New York 1990), Chapter 37,
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"Fluorescence In Situ Hybridization with DNA Probes," pp.3B3.
In addition to length determination, it is possible to
derive coarse base composition information from the DNA
strands. For example, the DNA specific dye Hoechst
preferentially binds to AT rich regions in DNA and
chromomycin preferentially binds to GC rich regions. By
exciting the fluorescence of these two dyes, as is done in
bivariate chromosome analysis (see Langlois, supra), the
AT:GC ratios for fragments can be determined. This ratio
will provide further fingerprinting information in addition
; to fragment length. Other binding dyes with different
sequence specificities can be used for fragment base
characterization. Further, synthesis of a piece of DNA in
the presence of fluorescently tagged nucleotide precursors
will label the piece of DNA according to its base
composition and this information can be subsequently
associated with the fragment length fluorescence analysis.
For example, replication of a piece of DNA using three
normal nucleotides and one fluorescently tagged C
nucleotide would then yield information about the number of
G nucleotides in the original piece since the tagged C
~ nucleotides would bind only to G nucletides.
.` An additional capability of the system can be provided
by using various sorting systems associated with flow
cytometers. See, e.g., U.S. Patent 3,710,933, supra, and
Flow Cytometry and Sorting, supra. A sorting capability
would enable one or more fragment sizes to be sorted from
`~ the flow stream for additional processing or study. If
. 30 hybridization probes are used, the sorting can separate the
hybridized fragments from the flow stream.
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Conventional sorting apparatus, as discussed in U.S.
Patent 3,710,933 and in T. Lindmo, "Flow Sorters for
Biological Cells,~ Flow Cytometry and Sortinq Second
Edition, Ed. M. Melamed et al., pp. 145-169, John Wiley &
Sons (1990), uses the fluorescence output signals discussed
above. After the fragments have passed through the
excitation volume for generating the output, the
hydrodynamic flow stream is broken into droplets by, e.g.,
ultrasonic vibrations, where each drop contains no more
than one fragment. Drops containing DNA fragments that
have a selected fluorescence response to an excitation are
charged by the application of a high voltage pulse across
the drops, which then pass through charged plates that
generate an electrostatic field to selectively deflect the
; 15 charged drops. The charge applied to the selected drops is
; controlled by circuitry that is responsive to fluorescent
emissions from the excitation volume within the flow
. cytometer, where the charging pulse is activated to produce
a deflection of drops containing a material emitting
fluorescence at a selected wavelength and intensity.
While the above description has been directed to DNA
pieces, the process is equally applicable to RNA strands.
Any reference to DNA in this case should be construed to
include RNA. Likewise, the form of signal detected is
taught to be fluorescence. However, any form of light
emission may be obtained, depending on the specific dye,
such that the term fluorescence should be interpreted to
include phosphorescence and luminescence. Further, the DNA
, or RNA being fingerprinted may not necessarily be from
humans, since all organisms have a genome that determines
their specific characteristics.
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The foregoing description of the preferred embodiments
of the invention have been presented for purposes of
illustration and description. It is not intended to be
exhaustive or to limit the invention to the precise form
disclosed, and obviously many modifications and variations
are possible in light of the above teaching. The
embodiments were chosen and described in order to best
explain the principles of the invention and its practical
application to thereby enable others skilled in the art to
~, 10 best utilize the invention in various embodiments and with
various modifications as are suited to the particular use
contemplated. It is intended that the scope of the
~ invention be defined by the claims appended hereto.
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