Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PURIFICATION AND DETECTION PROCESSES USING REVERSIBLE
AFFINITY ELECTROPHORESIS
RELATED APPLICATIONS
This application claims the benefit of U.S.
Provisional Application No. 60/076,614, filed on March 3,
1998, entitled "Purification and Detection Processes Using
Reversible Affinity Electrophoresis," the entire teachings
of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Zonal electrophoresis, and particularly gel
electrophoresis, is one of the best known methods for
separation, purification and characterization of charged
molecules, particularly macromolecules such as proteins or
nucleic acids (Freifelder, Physical Biochemistry, 2nd ed.,
(1982) pp. 276-310, Freeman, San Francisco).
Electrophoresis can be used to separate molecules based on
their size, charge, conformation, and many combinations of
these properties.
In most electrophoresis applications, charged
molecules migrate through a supporting medium under the
influence of an electric field. Most frequently,
electrophoresis is carried out using a linear constant
voltage gradient of fixed orientation (two fixed
electrodes, constant voltage). However, for very large DNA
molecules (i.e., in the size range of 30 to 2000 kb), the
polymeric chain orients with the field and snakes through
the gel rendering the sieving action of the electrophoretic
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medium ineffective. In order to separate large DNA
molecules, workers have developed applications in which the
field orientation is varied cyclically, as in "field
inversion gel electrophoresis" (Carle, et al., Science
(1986), 232:65) or "pulsed field" gel applications
(Schwartz and Cantor, Cell (1984), 37:67). Another
technique is to apply a constant field in a cyclic pulsed
fashion. Finally, approaches that combine both alternating
field and pulsed field duration have been described
(Bio-Rad Life Science Research Products Catalog (1997), pp.
175-182).
In most electrophoresis applications, the supporting
medium acts to suppress convection and diffusion, and can
be sieving or nonsieving. In affinity electrophoresis, the
support medium is .also modified with chemical groups (i.e.,
ligands) that interact specifically or nonspecifically with
one or more desired analytes and, thus, help to accomplish
the separation of analyte and non-analyte sample components
during purification by influencing its mobility.
Affinity electrophoresis has been used to measure the
binding affinity of proteins (Horejsi and Kocourek,
Biochim. Biophys. Acta (1974), 336:338-343 and Chu et al.,
J. Med. Chemistry (1992), 35:2915-2917). In addition,
vinyl-adenine modified polyacrylmide media has been used to
enhance resolution of nucleic acids in capillary
electrophoresis (Baba et al., Analytical Chemistry (1992),
64:1920-1924).
While many advances have been made in the resolving
power of electrophoresis, many biological macromolecules
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that contain only slight structural differences, for
example, a point mutation in a protein or nucleic acid,
still cannot be successfully separated. Analytical
techniques that improve resolution of biological molecules
are needed to provide researchers with the opportunity to
further probe and understand biological systems.
SUMMARY OF THE INVENTION
An affinity electrophoresis process is described, in
which the direction of the electric field is varied in a
cyclical manner while synchronously changing one, or more,
properties of the electrophoretic medium between two
states. In the first state, the property or properties
which are being varied favor specific reversible binding of
sample analytes to affinity ligands which are immobilized
within the medium. In the second state, the property or
properties which are being varied disfavor the binding of
sample analytes to the immobilized affinity ligands. The
process provides a convenient method to obtain high
resolution separations.
In another embodiment,.an apparatus for separating a
target analyte from a test sample is described. The
apparatus combines an electrophoretic medium having an
immobilized affinity ligand, an electrode system having
one, or more, electrodes, capable of generating an electric
field which can change in orientation, and a means of
changing one, or more, properties of the electrophoretic
medium between two states. In the first state, the
property or properties which are being varied favor
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specific reversible binding of sample analytes to affinity
ligands which are immobilized within the medium. In the
second state, the property or properties which are being
varied disfavor the binding of sample analytes to the
immobilized affinity ligands. The means of changing a
property in the electrophoretic medium can be a device
which changes the temperature of the electrophoretic
medium. Alternatively, the means of changing one, or more,
properties of the electrophoretic medium can be manually or
automatically changing the electrophoresis buffer.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA, 1B, and 1C show the separation of 5'-
Fluorescein-TGA GGC TTT CTG TTA TGG TAC-3' (SEQ ID N0: 1)
on an electrophoretic medium having a covalently bound
complementary nucleic acid strand from a non-complementary,
fluorescently labeled nucleic acid.
Figures 2A, 2B, 2C and 2D show the separation of E.
coli Rnase P RNA from 16S Hha RNA and 16S Alu RNA on an
electrophoretic medium having a covalently bound nucleic
acid sequence that is complementary to a sequence in E.
coli Rnase P RNA.
Figures 3A and 3B show the separation of E. coli Rnase
P RNA from 16S Hha RNA, 16S Alu RNA and total unlabeled RNA
from E. coli on an electrophoretic medium having a
covalently bound nucleic acid sequence that is
complementary to a sequence in E. coli Rnase P RNA.
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DETAILED DESCRIPTION OF THE INVENTION
The invention disclosed herein is directed to an
electrophoretic process of separating sample components,
and an apparatus designed to carry out the process, that
combines the following features: 1) an electrophoretic
medium that contains one or more immobilized affinity
ligands; 2) use of an electric field that changes in
orientation at least once during the process; and 3) a
change in at least one other medium property that affects
the ability of the affinity ligand(s) to form a specific
binding complex with the analyte{s), said change in medium
property (or properties) occurring synchronously with the
change in field orientation, thereby allowing
electrophoretic separation of analyte and non-analyte
components of the sample.
The combination of these features results in a novel
invention that has broad utility for separation,
purification and detection of molecules, including
proteins, nucleic acids, and other charged species.
In one preferred embodiment of the invention, the
process is a general method for performing repetitive
cycles of affinity separation for purification of specific
analytes in a biological or test sample. In this
embodiment, the analyte molecules are purified (e. g.,
isolated or separated) from non-analyte sample components.
Each cycle is characterized by two electrophoretic steps.
The first step is carried out using a first field
orientation and first medium condition (also referred to
herein as "state"), said condition allowing formation of
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specific binding complexes between sample analytes and
affinity ligands in the medium. Under those conditions,
only non-analyte sample components will migrate in the
medium, allowing fractionation of the sample based on
analyte affinity for the ligand. The next step is carried
out under a second field direction and second medium
condition which is obtained by varying one or more property
of the electrophoretic medium from the first medium
condition. The second condition is designed to disrupt
formation of specific binding complexes between sample
analytes and affinity ligands in the medium. During this
step, all sample components are moved to new locations
within the medium. At the completion of this step partial,
or complete, separation of specific analytes from other
sample components has occurred, and both fractions have
been moved to new locations within the medium.
For some sample/analyte/ligand combinations, a single
cycle may provide sufficient purification of analyte for
many applications. If additional purification is desired,
the purification cycle can be repeated. For example,
during the second cycle, purification proceeds as in the
first cycle. However, the starting materials for the second
purification cycle are the partially fractionated products
of the first purification cycle which are now at new
locations in the electrophoretic medium. The locations of
analyte and non-analyte fractions from the first cycle may
or may not overlap, depending on the extent of purification
achieved in the previous cycle. In either case, during the
second and subsequent cycles, the two fractions are further
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separated. Thus, an arbitrarily high number of affinity
purification cycles can be performed on a single
electrophoresis unit, in an automated fashion, with
continuous removal of non-binding sample components during
each cycle. In systems in which there is tight binding
between a gel-immobilized affinity ligand and an analyte
and low non-specific binding of non-target molecules for
the affinity ligand, a low number of cycles, for example 1
to 10 cycles, may provide the necessary purification. In
other cases, where ligand-target binding is weak, or where
there is significant binding of non-target molecules to the
affinity ligand, many cycles, for example 10 to several
thousand, may be required to separate the components.
The repetitive nature of the process allows extremely
efficient electrophoretic purification of the analyte
molecules. For instance, if the purification efficiency of
each cycle is 10-fold (e. g., 90~ of non-analyte sample
components removed per cycle), four cycles would yield a
purification of 10,000-fold.
A key advantage of the invention is that the cyclic
purification process can be carried out in a single device,
such as an electrophoretic gel. This simplifies the
purification process by eliminating preparation, loading,
and fraction collection from multiple columns.
In addition, for applications involving simple changes
of state, such as temperature, to control ligand/analyte
interactions, the process can be performed in an automated
fashion using equipment with few (or no) moving parts.
Discussions of the key components of the invention and
nonlimiting extensions are listed below:
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TEST SAMPLE
The test sample can be any sample, from any source in
which analyte molecules are mixed with non-analyte
molecules. An analyte molecule is any molecule of interest
that can form a binding complex with an affinity ligand.
Specifically encompassed by the present invention are
samples from biological sources containing cells, obtained
using known techniques, from body tissue (e. g., skin, hair,
internal organs), or body fluids (e. g., blood, plasma,
urine, semen, sweat). Other sources of samples suitable
for analysis by the methods of the present invention are
microbiological samples, such as viruses, yeasts and
bacteria: plasmids, isolated nucleic acids and agricultural
sources, such as recombinant plants.
The test sample is treated in such a manner, known to
those of skill in the art, so as to render the analyte
molecules contained in the test sample available for
binding. For example, a cell lysate can be prepared, and
the crude cell lysate (e. g., containing the target analyte
as well as other cellular components) can be analyzed.
Alternatively, the target analyte can be partially isolated
(rendering the target analyte substantially free from other
cellular components) prior to analysis. Partial isolation
can be accomplished using known laboratory techniques. For
example, DNA, RNA and proteins can be isolated from a
variety of biological samples using TRI reagent (see Sigma
catalogue, p. 1545, catalogue numbers T9424, T3809, and
T3934, see also Chomczynski, et al., Biochem. (1987),
162:156; Chomczynski, Biotechniques (1993), 15:532) in
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conjunct with Southern blotting (DNA), Northern blotting
(RNA) and Western blotting (proteins) procedures.
Antibodies can be isolated by binding to Protein A
immobilized on a solid support (see Surolia, et al., Trends
Bioch. Sci. (1981), 7:74 and Sigma catalogue p. 1462,
catalogue number PURE-1). A nucleic acid analyte can also
be amplified (e. g., by polymerase chain reaction or ligase
chain reaction techniques) prior to analysis.
AFFINITY LTGANDS THAT BIND REVERSIBLY TO AN ANALYTE
An affinity ligand is any molecule that can form a
specific binding complex with an analyte and can be
immobilized within a suitable electrophoretic medium.
Methods for determining the thermal stability of
binding complexes and, in particular, hybridization
complexes are well known in the literature. Wetmur,
Critical Reviews in Biochemistry and Molecular Biology,
26:227-259 (1991); Quartin and Wetmur, Biochemistry,
28:1040-1047 (1989). Application of these methods to
estimate the stability of an analyte/affinity ligand
complex concerns the following reaction:
k2
D + D' B
kr
wherein D and D' are an affinity ligand and an analyte,
such as a first nucleic acid and a second nucleic acid
containing a region complementary to the first nucleic acid
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sequence, B is the analyte/affinity ligand complex product
and kz and kr are the kinetic rate constants for the
analyte/affinity ligand complex formation and dissociation,
respectively. In this scheme, the reverse reaction is most
relevant to the consideration of spontaneous dissociation
of the analyte/affinity ligand complex, and the rate
constant for dissociation, kr, is the critical variable
that needs to be minimized to facilitate binding between
the analyte and affinity ligand. For a given
analyte/affinity ligand complex, dissociation can be
reduced by lowering the assay temperature; this will
decrease the dissociation constant.
Once a measurement of the dissociation constant has
been obtained for one experimental temperature, the
Arrhenius equation (2) can be rearranged to calculate the
kr for other temperatures as follows:
k = A exp (-Ea/RT) (2)
krl/kr2 = exp C (Ea/R) f 1/TZ) - (1/T,) }1 (3)
wherein krl and kr2 are the analyte/affinity ligand complex
dissociation rate constants at temperature T1 and T2, Ea is
the activation energy for dissociation and R is the
universal gas constant. For a nucleic acid
analyte/affinity ligand complex, the term Ea can be
calculated from the base sequence of the nucleic acid
sequence used to form the analyte/affinity ligand complex.
Wetmur, Critical Reviews in Biochemistry and Molecular
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Biology, 26:227-259 (1991). Use of the Arrhenius equation
for this calculation is described by Tinocco, et al.,
Physical Chemistry: Principles and Applications in
Biological Sciences, Prentice Hall (pub.), Englewood
Cliffs, NJ, pp. 290-294 (1978) .
In the case where the analyte/affinity ligand binding
reactions occur in discrete regions of a solid support
matrix, such as an electrophoresis matrix, an effective
dissociation constant can be estimated using a temperature
l0 gradient procedure. The melting behavior of an immobilized
analyte/affinity ligand complex within an electrophoresis
gel can be measured using a temperature gradient which
increases laterally across the gel. The temperature, Td,
at which 50% of the complex has dissociated during the time
of electrophoresis, ta, can be used to estimate the
dissociation constant.
Considering the dissociation as a first order reaction
with kinetic rate constant kr, it follows that at Td:
ln(0.5) - -krta (4)
kr = -0.693/ta (5)
Thus, using temperature gradient gels allows for the
measurement of an effective value for kr, Td and ta. Once
kr has been evaluated at Td, equation (3) can be used to
calculate kT at other lower temperatures that might be
suitable for the first medium condition wherein conditions
are selected to allow the formation of specific binding
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complexes between sample analytes and affinity ligands.
These calculated values of kr can then be used with the
first order rate law to calculate the fraction of
analyte/affinity ligand complex remaining at a given assay
temperature to and electrophoresis time ta:
ln(B/Bo) - -krta (6)
wherein B is the concentration of analyte/affinity ligand
complex remaining at time ta, and Bo is the initial
concentration of the complex. Equation (6) can be used to
estimate the change in kr needed to increase B/Bo (decrease
analyte/affinity ligand complex dissociation) by any
specified amount. Once the desired value of kr is known,
equation (3) can be used to calculate the change in
temperature needed to achieve the kr value.
It should be noted that the gradient gel procedure
only provides an estimate of the actual analyte/affinity
ligand complex Td and kr, since displaced analytes can
rebind to uncomplexed immobilized affinity ligands. In
general, the experimentally determined values will
overestimate the actual Td and underestimate the actual kr
for the reversible analyte/affinity ligand complex
dissociation reactions. Nevertheless, the quantitative
relationships given in equations (1) through (6) provide a
rational and practical framework for predicting the
stability of analyte/affinity ligand complexes, and design
of medium conditions for separation protocols.
One especially useful example of an affinity ligand is
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a single-stranded nucleic acid, which can bind by
hybridization, for example, to an analyte that contains a
complementary nucleic acid sequence. The single strand
nucleic acid affinity ligand can be complementary to the
entire analyte nucleic acid sequence or to a portion
thereof. Single-stranded nucleic acids can also be used for
isolation of duplex nucleic acids by triplex formation
(Hogan and Kessler, U.S. Patent No. 5,176,966 and Cantor,
et al., U.S. Patent No. 5,482,836, the teachings of which
are incorporated herein by reference). Double-stranded
nucleic acids can also serve as useful affinity ligands for
nucleic acid binding proteins, or for nucleic acid analytes
that bind to the ligand by triplex or tetraplex formation.
The conditions under which a single strand nucleic
acid will bind to another nucleic acid to be immobilized in
a gel can be estimated using the procedure outlined above
for estimating the stability of analyte/affinity ligand
complexes. In addition, the melting temperature (Tm) of
the two nucleic acids provides a reasona$le framework for
estimating the temperate at which an nucleic acid analyte
will hybridize to a nucleic acid affinity ligand. In
general, the Td is lower than the Tm by about 15 to 25°C
and, therefore, the temperature at which the gel should be
run to facilitate specific hybridization between the
analyte and affinity ligand should be about 15 to 25°C or
more below the Tm.
The Tm of a pair of nucleic acids is typically
determined by monitoring a physical property, such as UV
absorption, of a solution of the two nucleic acids in the
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electrophoresis buffer while uniformly varying the property
of the solution that will be cyclically varied during the
electrophoresis separation. For example, the temperature
can be slowly decreased while monitoring the UV absorption.
At high temperatures the nucleic acids are single stranded.
As the temperature decreases complementary bases pair off
and hydrogen bond. This hydrogen bonding causes a change
in UV absorption. If the nucleic acids are complementary,
the transition between the hydrogen bonding state and the
non-hydrogen bonding state occurs over a narrow temperature
range. The midpoint of this temperature range is the Tm
for the two nucleic acids. Similarly, the ionic strength
or pH of the buffer can be varied in a uniform manner while
holding the temperature constant and monitoring the W
absorption.
Nucleic acids form duplexes more readily in higher
ionic strength and lower temperature conditions.
"Stringency conditions" for hybridization is a term of art
which refers to the conditions of temperature and buffer
concentration (ionic strength) which permit hybridization
of a particular nucleic acid to a second nucleic acid in
which the first nucleic acid may be perfectly complementary
to the second, or the first and second may share some
degree of complementarity which is less than perfect. For
example, certain high stringency conditions can be used
which distinguish perfectly complementary nucleic acids
from those of less complementarity. "High stringency
conditions" and "moderate stringency conditions" for
nucleic acid hybridizations are explained on pages 2.10.1-
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2.10.16 (see particularly 2.10.8-11) and pages 6.3.1-6 in
Current Protocols in Molecular Biology (Ausubel, F.M. et
al., eds., Vol. 1, containing supplements up through
Supplement 29, 1995), the teachings of vihich are hereby
incorporated by reference. The exact conditions which
determine the stringency of hybridization depend not only
on ionic strength, temperature and the concentration of
destabilizing agents such as formamide, but also on factors
such as the length of the nucleic acid sequence, base
composition, percent mismatch between hybridizing sequences
and the frequency of occurrence of subsets of that sequence
within other non-identical sequences. Thus, high or
moderate stringency conditions can be determined
empirically.
By varying hybridization conditions from a level of
stringency at which no hybridization occurs to a level at
which hybridization is first observed, conditions which
will allow a given sequence to hybridize (e. g.,
selectively) with the most similar sequences in the sample
can be determined. Binding conditions for triplexes and
tetraplexes can be estimated in a similar manner.
Nucleic acid aptamers (Tuerk and Gold, Science (1990)
249:5050; Joyce, Gene (1989), 82:83-87; Ellington and
Szostak, Nature (1990), 346:818-822) can also be used as
affinity ligands in the process of the present invention.
Aptamers can be selected against many kinds of analytes,
including proteins, small organic molecules, and
carbohydrates (reviewed in Klug and Famulok, Molecular
Biology Reports (1994), 20:97-107). Thus, selection of
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aptamer ligands offers a simple and flexible mechanism for
obtaining affinity ligands against virtually any target
molecule.
Other useful ligands include proteins or polypeptides
which can bind to specific analytes. An especially useful
class of protein ligands are antibody molecules, which can
be elicited against a wide range of analytes by
immunization methods. Antibodies ligands can be monoclonal
or polyclonal. In addition, a fragment of an antibody can
be an affinity ligand. Similarly, receptor proteins may be
useful as ligands for purification and detection of
analytes that bind to or are bound by them.
Carbohydrates have been successfully used as affinity
ligands for electrophoretic purification of lectins
(Horejsi and Kocourek, Biochim. Biophys. Acta (1974),
336:338-343), and may be useful for purification and
detection of molecules that bind to specific carbohydrates
or glycoproteins.
Binding or non-binding conditions of proteins,
aptamers and lectins for specific ligands can be estimated
using the procedure outlined above for estimating the
stability of analyte/affinity ligand complexes. In
addition, equilibrium dialysis experiments can provide a
rational method of predicting the stability of
analyte/affinity ligand complexes. For example, the
dissociation constant of a protein for a particular ligand
can be determined in the electrophoresis buffer at several
different pHs, temperatures or ionic strengths. The higher
the dissociation constant, the weaker the binding between
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the protein and the ligand (see Segel, I.H., Biochemical
Calculations, 2nd Edition (1976), John Wiley & Sons, N.Y.,
p. 241-244). From this data a binding and a non-binding
condition can be estimated.
Many other types of immobilized ligands are possible
including peptides, amino acids, nucleosides, small organic
molecules, lipids, hormones, drugs, enzyme substrates,
enzyme inhibitors, enzymes, coenzymes, inorganic molecules,
chelating agents, macromolecular complexes,
polysaccharides, monosaccharides, and others.
AN ELECTROPHORETIC MEDIUM WHICH CONTAINS AT LEAST ONE
IMMOBILIZED AFFINITY LIGAND
Any medium suitable for electrophoresis can be used
for the methods of the present invention. In general,
suitable media fall into two classes. The first includes
media composed of gel-forming materials like crosslinked
polyacrylamide and agarose. The second class includes
media composed of solutions of linear noncrosslinked
polymers such as polyacrylamide,
poly(hydroxyethylcellulose), and poly(ethyleneoxide). The
latter category is commonly used for capillary
electrophoresis applications.
Immobilization of ligands can be accomplished by
direct attachment to the polymeric components of the
medium. Such attachment can be mediated by formation of
covalent bonds between the ligand and the polymer.
Noncovalent binding between the ligand and polymer
substituents can also be used. For instance, strong
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noncovalent binding provided by the widely-used
biotinstreptavidin and digoxigenin-antidigoxigenin systems
can be used to attach ligands to appropriately modified
polymeric media. Covalent attachment is preferred.
Direct connection between the polymeric medium and the
ligand is not strictly required. For instance, ligands can
be attached to particulate supports, such as microspheres,
and the particulate supports can be immobilized within the
polymer medium by physical entrapment (Cantor, et al., U.S.
Patent No. 5,482,863, r_he teachings of which are
incorporated herein by reference in their entirety). The
particles may be macroscopic, microscopic, or colloidal in
nature, (see Polyciences, Inc., 1995-1996 particle Catalog,
Warrington, PA).
In a similar manner, ligands can be attached to highly
branched soluble polymers. Due to their branched shape,
such ligand-polymer complexes display extremely large
effective hydrodynamic radii and, therefore, will not
migrate in the electric field in many kinds of polymeric
media of appropriately small pore size. Thus, they can be
entrapped within the media in the same fashion as
particulate supports.
Absolute immobilization of the ligand within the
medium is not required for all embodiments of the
invention. Fcr many applications, it is sufficient that
the mobility of the analyte is changed upon formation of a
binding complex with the ligand. This condition can be
satisfied by coupling the ligand to a medium component that
has extremely low electrophoretic mobility. However, for
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efficient purification the change in mobility should be as
large as possible. Therefore, media utilizing true
immobilization of the ligand within the medium will be
preferred for use in this invention.
Commonly used gel media useful for the present
invention include acrylamide and agarose gels. However,
other materials may be used. Examples include modified
acrylamides and acrylate esters (for examples see
Polysciences, Inc., Polymer & Monomer catalog, 1996-1997,
Warrington, PA), starch (Smithies, Biochem. J. (1959),
71:585; product number 55651, Sigma Chemical Co., St.
Louis, MO), dextrans (for examples see Polysciences, Inc.,
Polymer & Monomer Catalog, 1996-1997, Warrington, PA), and
cellulose-based polymers (for examples see Quesada, Current
Opinions in Biotechnology (1997), 8:82-93). Any of these
polymers can be chemically modified to allow specific
attachment of ligands (including nucleic acids, proteins,
peptides, organic small molecules, and others) for use in
the present invention.
For some methods, it may be useful to use composite
media, containing a mixture of two or more supporting
materials. An example is the composite acrylamide-agarose
gel. These gels typically contain from 2-5% acrylamide and
0.5%-1% agarose. In these gels the acrylamide provides the
chief sieving function, but without the agarose, such low
concentration acrylamide gels lack mechanical strength for
convenient handling. The agarose provides mechanical
support without significantly altering the sieving
properties of the acrylamide. In such cases, the nucleic
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acid can be attached to the component that confers the
sieving function of the gel, since that component makes
most intimate contacts with the solution phase nucleic acid
target.
For capillary electrophoresis (CE) applications it is
convenient to use media containing soluble polymers.
Examples of soluble polymers that have proven to be useful
for CE analyses are linear polymers of polyacrylamide,
poly(N,N-dimethylacrylamide), poly(hydroxyethylcellulose),
poly(ethyleneoxide) and poly(vinylalcohol) as described in
Quesada, Current Opinion in Biotechnology (1997), 8:82-93).
Solutions of these polymers can also be used to practice
the methods of the present invention.
Methods of coupling a variety of ligands to create
affinity electrophoresis media are well known to those
skilled in the art. Many ligands can be coupled to
agarose, dextrans, cellulose, and starch polymers using
cyanogen bromide or cyanuric chloride activation. Polymers
containing carboxyl groups can be coupled to ligands that
have primary amine groups using carbodiimide coupling.
Polymers carrying primary amines can be coupled to
aminecontaining ligands with glutaraldehyde or cyanuric
chloride. Many polymers can be modified with
thiol-reactive groups which can be coupled to
thiol-containing ligands. Many other suitable methods are
known in the literature. For examples, see Wong,
"Chemistry of Protein Conjugation and Cross-linking", CRC
Press, Boca Raton, FL, 1993.
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Methods for covalently attaching ligands by
copolymerization with the polymeric material of the
electrophoretic medium have also been developed. In this
approach, ligands are chemically modified with a
copolymerizable group. When such modified ligands are
copolymerized with suitable mixtures of polymerizable
monomers, polymeric media containing high concentrations of
immobilized ligand can be produced. Examples of methods
for covalently attaching nucleic acids to polymerizable
chemical groups are found in U.S. Patent Application Serial
No. 08/812,105, entitled "Nucleic Acid-Containing
Polymerizable Complex," and U.S. Patent Application Serial
No. 08/971,845, entitled "Electrophoretic Analysis of
Molecules Using Immobilized Probes," the teachings of which
are herein incorporated by reference, in their entirety.
(See also, Rehman, et al., Nucleic Acids Research (1999),
27:649.) Other useful methods that have been used to
immobilize proteins and small organic molecules within
polymer layers and gels are described in Bille et al., Eur.
J. Biochem. (1989), 180:41-47; Wang et al., Nature
Biotechnology (1997), 15:789-793; and Holtz and Asher,
Nature (1997), 389:829-832.
Other approaches for attaching nucleic acid probes to
preformed polyacrylamide polymers, including gels or linear
soluble polymers can be found in Ghosh and Fahy, U.S.
Patent No. 5,478,893, the teachings of which are
incorporated herein by reference in their entirety, and in
Timofeev et al., Nucleic Acids Res. (1996), 24:3142-3148.
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PROCESS AND MEANS FOR VARYING ORIENTATION OF
ELECTROPHORETIC FIELD DURING SEPARATION
An electrode system is a system that, in conjunct with
a power supply, produces and electric field gradient. An
electric field gradient is the voltage drop across the gel
created by the electrode system (see Giddings, Unified
Separation Science (1991), John Wiley & Sons, New York, p.
155-170). The orientation of the electric field gradient
used for electrophoresis determines the geometry of the
separation between analyte and non-analyte sample
components. Many field geometries can be used. For
instance, with a conventional two-electrode apparatus, a
one-dimensional separation can be achieved simply by
switching the polarity of the two electrodes, as practiced
in field inversion gel electrophoresis (Carle et al.,
Science (1986), 232:65). Ir~ separations of this sort,
analytes only migrate under conditions which disfavor
binding to the ligands, whereas the non-analyte sample
components would migrate under both sets of conditions.
For purposes of illustration, let the direction of net
analyte movement during the purification process be called
"forward". If the purification cycle is designed so that
the duration of reverse field orientation is longer than
the duration of forward field orientation, analyte
molecules will be moved forward during each purification
cycle, but non-analyte sample components will only enter
the gel only transiently since they are efficiently removed
from the gel by the long period of reverse field
orientation.
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Two dimensional electrode arrangements, as used in
pulsed field (Schwartz and Cantor, CeI1 (1984), 37:67) and
CHEF applications (CHEF gels, U.S. Patent No. 5,549,796;
Bio-Rad Life Science Research Products Catalog (1997), pp.
175-182), allow the separation process of the present
invention to be performed in two dimensions. In principle,
the addition of another set of electrodes operating in a
third dimension could add additional separation capability
if desired.
The state of instrumentation and methodology for
performing one and two dimensional electrophoretic
separations is well advanced. At least one commercially
available device (CHEF gel apparatus, Bio-Rad Life Science
Research Products Catalog, 1997, pp. 175-182), offers the
capability of performing two-dimensional electrophoretic
separations with programmable automated control of field
orientation and pulse duration.
PROCESS AND MEANS FOR VARYING AT LEAST ONE OTHER PROPERTY
OF THE ELECTROPHORETIC MEDIUM IN SYNCHRONY WITH THE CHANGE
IN ELECTROPHORETIC FIELD ORIENTATION
The electrophoretic medium can be reversibly cycled
between at least two different user-defined states by
varying one or more property of the electrophoretic medium
(e. g., temperature, pH or ionic strength) . In one state,
the ligand has a relatively high affinity for the analyte
of interest. In the other state, the ligand has relatively
low binding affinity with the analyte. In a preferred
embodiment, non-analyte sample components have low affinity
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for the ligand in both states. In a more preferred
embodiment of the invention, variation in medium state and
the orientation of the electric field are co-regulated, so
that electrophoresis of non-analyte materials occurs under
all conditions and field orientations, but electrophoresis
of analytes occurs only under a limited set of conditions
and field orientations. Thus, analytes and non-analyte
molecules will have different net mobilities for each
cycle, and their separation in the medium will increase
with each cycle.
Changing the medium temperature is one preferred means
for modulating analyte-ligand binding affinity, since
temperature can be varied with little or no manipulation of
the electrophoresis medium, and since a great deal of
instrumentation for temperature control is commercially
available. However, other medium properties may be used as
well. A non-limiting list of possible properties which are
known to affect noncovalent chemical associations include
changes in medium pH, changes in the ionic strength of the
medium, and other changes in chemical composition of
medium.
In one especially preferred embodiment of the
invention, the affinity ligand is an nucleic acid and the
analyte is a sample nucleic acid that has at least one
region complementary to the affinity ligand nucleic acid.
In this case, the binding between analyte and ligand can be
effectively modulated by changing the gel temperature. For
example, at temperatures above the Td of the ligand-analyte
complex, binding affinity will be low. Similarly, at
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temperatures below the Td, binding affinity will be
substantially higher.
Processes and means for cycling electrophoretic media
between two temperatures are well known to those skilled in
the art. For example, temperature- controlled equipment
for performing vertical or horizontal format
electrophoresis are commercially available (Bio-Rad Life
Science Research Products Catalog (1997), pp. 127-133,
175-182; Pharmacia Biotech BioDirectory (1997), pp. 345,
309, 334). In some instruments, temperature control is
achieved by circulation of water (or suitable coolant)
through the instrument. In these instruments, temperature
cycling can be achieved by the switching coolant source
between two regulated reservoirs set at the desired
temperatures. In some electrophoresis instruments, the
medium is in thermal contact with a programmable
thermocycler which relies on the Petier effect for heating
and cooling. (Thermocyclers can be obtained from MJ
Research, watertown MA). For example, and electrophoresis
unit with a Peltier heating/cooling devise can be obtained
from Pharmacia (Pharmacia Biotech BioDirectory {1997), pp.
334).
Another method of modulating the analyte-ligand
binding affinity is by changing the ionic strength of the
electrophoresis buffer. The ionic strength of the buffer
that will facilitate binding is dependent on the type of
analyte and the affinity ligand. In general, a buffer that
has a higher ionic strength facilitates binding. Buffers
that have ionic strengths of about 100 mM to 1 M are
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preferred during the state in which the analyte is bound to
the affinity ligand. Buffers that have ionic strengths of
about 10 mM or less are preferred during the state in which
the analyte is not bound to the affinity ligand.
Equilibrium dialysis or hybridization experiments can be
used to provide a rational for predicting the stability of
a particular analyte/affinity ligand binding complex at a
particular ionic strength.
Another method of modulating the analyte-ligand
binding affinity is by changing the electrophoresis buffer
to a denaturing buffer. A denaturing buffer contains
chemicals (hereinafter "denaturants") which disrupt the
binding of the analyte to the affinity ligand. For
example, formamide or urea can be a component of the
denaturing buffer. The amount of denaturant required will
depend on the type of target molecule, the type of affinity
binding interaction, field strength, ionic strength, and
temperature of electrophoresis. In general, the denaturing
buffer can have a very broad concentration range of
formamide or urea. Formamide can be used in concentrations
up to 95~ (volume/volume), and urea can be used at
concentrations up to 8M. Equilibrium dialysis or
hybridization experiments can also be used to provide a
rational for predicting the stability of the
analyte/affinity ligand binding complex in a particular
denaturing buffer.
In some instances, the analyte-ligand binding affinity
can also be modulated by changing the pH of the buffer.
For example, nucleic acids will hybridize to a
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complementary nucleic acid affinity ligand at or near
neutral pH (e. g., pH 6-8). DNA affinity ligands hybridized
to DNA targets can be disrupted by acidic pH (e. g., below
pH 5) or basic pH (e.g., above pH 11). Changing the pH of
the electrophoresis buffer is a preferred method of
disrupting the binding of protein analytes to an affinity
ligand. Equilibrium dialysis experiments can be used to
estimate the pH range for binding and dissociation of a
particular protein to an analyte.
For maximum separation efficiency, switching medium
conditions should switch the analyte between completely
bound and completely unbound states. This clean
distinction between bound and unbound analyte states can be
achieved with single-stranded nucleic acid analyte-ligand
systems, as exemplified in later sections of this
disclosure. However, such absolute two-state behavior is
not required for successful application of the invention.
In general, it is sufficient that the mobility of the
analyte be substantially altered by the change in medium
conditions. Here "substantially" means that the mobility
change observed in a ligand-containing medium is greater
than the mobility change observed for a similar medium
lacking the ligand, or alternatively, a medium containing a
ligand which is chemically similar to the original ligand
but which is incapable of forming specific binding
complexes with analyte. Given such a substantial change in
analyte mobility, even weak specific ligand/analyte
interactions can be used successfully to practice the
present invention, since an arbitrarily large number of
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purification cycles can be repeated in an automated w
fashion. In these cases, each cycle gives a small but
finite separation between analyte and non-analyte sample
components, and the purification cycle is repeated until
the required level of separation is achieved.
APPLICATIONS OF THE INVENTION
The invention can be used to purify analytes for
subsequent characterization and other preparative purposes.
In addition, the invention can be used for detection of
analytes.
For preparative purposes, the invention is powerful
because it allows the potential for performing many
repetitive cycles of affinity purification using a single
automated programmable device with inexpensive, easy to
prepare affinity media. Preferably, elution of purified
product could be accomplished electrophoretically using a
variation of methods disclosed in Gombocz et a1. U.S.
Patent No. 5,217,591 and Kragt and Ballen, U.S. Patent No.
3,989,612, the teachings of which are incorporated herein
by reference in their entirety. Other elution methods are
possible and are well known to those skilled in the art.
For detection purposes, the invention is powerful
because it allows removal of non-analyte sample components
which can contribute to unfavorable levels of background.
The general format of these assays involves the following
steps:
1) preparation of sample to be tested;
2) purification of potential sample analyte by
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method of present invention; and
3) assay for analyte in output of purification
process in step 2.
As nonlimiting examples of useful detection applications
using the invention, three variations of this general
scheme are presented below. The examples are not intended
to limit the scope of the invention in any way. Other
detection methods may be more useful for other types of
analyte. All are directed toward detection of bacteria in a
blood sample.
a) Detection by dye binding
In step 1, total RNA is prepared from the blood sample
(Chomczynski, et al., Anal. Biochem. (1987), 162:156;
Chomczynski, Biotechniques (1993), 15:532; TRI Reagent BD,
Sigma {1999), catalog no. T3809, p. 1545). In step 2, RNA
is subjected to the method of the present invention using
an affinity medium specific for binding bacterial ribosomal
RNA. In step three, the output of the purification process
is tested for the presence of RNA using dyes which
fluoresce brightly when bound to nucleic acids, for
example, Acridine Orange, SYBR green II, TO-TO or YO-YO.
Other examples of suitable dyes can be found in Haugland,
"Handbook of Fluorescent Probes and Research Chemicals, 6th
Edition," Molecular Probes, Eugene, OR, (1996), pp.
144-156.
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b) Detection by copurification of enzymatic label
In step 1, total RNA is prepared from the blood
sample, and the bacterial ribosomal RNA analyte within the
sample is bound to an enzymatic reporter molecule, such as
alkaline phosphatase, horseradish peroxidase, or luciferase
by means which are not disrupted during the purification
process of step 2. Preferably, attachment of the RNA
analyte to the~enzyme reporter is mediated by a nucleic
acid sandwich hybridization probe (Ranki and Soderland,
U.S. Patent No. 4,486,539; Engelhardt and Rabbani, U.S.
Patent No. 5,288,609, the teachings of which are
incorporated herein by reference in their entirety) to
which the enzyme is conjugated. The assay of step 3
detects the enzymatic reporter molecule using chromogenic,
chemifluorescent, or chemiluminescent substrates.
c) Detection by copurification of amplifiable reporter
label
In step 1, total RNA is prepared from the blood
sample, and the bacterial ribosomal RNA analyte within the
sample is bound to an amplifiable reporter molecule, such
as a substrate of Q-beta replicase, for example the MDV-1
substrate of Q-beta replicase (Lizardi et al.
Bio/Technology 6:1197-1202, 1988), by means which are not
disrupted during the purification process of step 2.
Preferably, attachment of the RNA analyte to the enzyme
reporter is mediated by a nucleic acid sandwich
hybridization probe, to which the amplifiable reporter is
conjugated. The assay of step 3 detects the amplifiable
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reporter molecule using an appropriate enzymatic
amplification and detection process.
It will be obvious to those skilled in the art that
the examples (a) through (c) above are listed in order of
increasing sensitivity. The theoretical sensitivity of
example (c) is limited only by loss of analyte and/or
amplifiable reporter during sample preparation and
electrophoretic purification, since the Q-beta replicase
system is capable of detecting a single MDV-1 substrate in
a sample.
The invention is illustrated by the following
examples, which are not intended to be limiting in any way.
EXAMPLES
Example 1. Separation of nucleic acid samples
An example of the present invention used to separate a
mixture of nucleic acids, is shown in Figure lA and 1B. In
this example, the ligand is an nucleic acid GTA CCA TAA CAG
CAA GCC TCA (SEQ ID NO: 2) covalently immobilized in an
standard polyacrylamide gel using the methods of U.S.
patent applications Serial Nos. 08/812,105 and 08/971,845,
the teachings of which are incorporated herein by reference
in their entirety.
A fluorescently labeled nucleic acid,
5'-Fluorescein-ATT ACG TTG ATA TTG CTG ATT A-3' (SEQ ID NO:
3), that is not complementary to the ligand (signified by a
box) was loaded in lane 1. A fluorescently labeled nucleic
acid, 5'-Fluorescein-TGA GGC TTT CTG TTA TGG TAC-3' (SEQ ID
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N0: 1), complementary to the ligand was loaded in lane 2.
This preparation contains a minor species (signified by an
open circle) which shows a slightly greater mobility than
the major species (signified by closed circles) for unknown
reasons. Lane 3 contained a mixture of the two
fluorescein-tagged nucleic acids.
Figures lA and 1B show the gel after the end of steps
1 and 2 in the first cycle. In step 1, the gel was
maintained at 45oC. At this temperature, binding of the
ligand to complementary nucleic acids was prevented, and
the electric field, applied for 43 minutes at 100 V, had
the polarity indicated in Figure 1. In step two, the gel
was maintained at 25oC, a temperature which allows binding
of the ligand to complementary nucleic acids. The electric
field, applied for 50 min at 100 V, had the opposite
polarity to that used in step 1. At the end of step one,
the mobility of the two fluorescently labeled nucleic acids
was similar, as shown in Figure lA. At the end of step
two, a separation of the two nucleic acids was observed
(Figure 1B). Figure 1C, shows the same gel after a total
of three cycles. Each cycle consisted of steps 1 and 2
(down/hot and up/cold) as described for Figures lA and 1B,
above. After the third cycle, the separation between the
complementary and noncomplementary nucleic acids was
increased. In particular, the complementary nucleic acid
moved further down the gel, while the noncomplementary
nucleic acid moved in the reverse direction.
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GENERATION OF RNA ANALYTES USED IN EXAMPLES 2 AND 3
Three RNA analytes were used for the experiments shown
in Figures 2 and 3. All three were generated by in vitro
transcription reactions using T7 polymerase (enzyme from
Boehringer Mannheim catalog #881,767; other reaction
components from Promega kit catalog #P1420) with
fluorescently tagged ribonucleotide triphosphates as label
(fluorescein labeled nucleotides from Boehringer Mannheim
catalog #1,685,61 9). The three RNA molecules were:
1. 16S Hha, 502 nucleotides in length, nonspecific sample
component.
2. 16S Alu, 255 nucleotides in length, nonspecific sample
component.
3. E. coli RNase P RNA, 377 nucleotides in length,
specific RNA analyte.
These RNA species were generated as detailed below:
16S Hha and 16S Alu
Plasmid pSCH038 was obtained from the American Type
Culture Collection (ATCC87435). The plasmid is a PCRII
plasmid vector (Invitrogen, Carlsbad, California, catalogue
number K2050-01) containing the E. coli 16S ribosomal RNA
(rRNA) sequence from positions 674 to 1411. The complete
16S molecule is 1541 nucleotides in length. The vector
contains a promoter site for T7 RNA polymerase to the 5'
side of insert so that in vitro transcription with that
enzyme will produce RNA with the same sense as native 165
rRNA.
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Two 5 mg aliquots of plasmid were digested with the
restriction enzymes Hha I or Alu I. The digested plasmid
samples were used as templates for in vitro transcription
with T7 polymerise and fluorescein-labeled nucleotides.
After synthesis the DNA templates were removed by digestion
with Dnase I, and the transcripts were purified from
unincorporated nucleotides using Pharmacia G25 spin
columns.
The first 68 nucleotides of both transcripts are
derived from the cloning vector PCRII: there are 68
nucleotides between the T7 initiation site and the 16S
insert. Alu I cleaves between nucleotides 860 and 861 of
the 16S rRNA sequence. Therefore, the length of the
transcript generated from the Alu 1 cleaved template, 16S
Alu, is 255 nucleotides.
Hha I cleaves the 16S sequence between nucleotides
1107 and 1108 of the 16S rRNA sequence. Therefore, the
length of the 16S Hha transcript is 502 nucleotides.
E. coli Rnase P RNA
Naturally occurring E. coli Rnase P RNA is 377
nucleotides in length (Altman, et al., In tRNA: Structure,
Biosynthesis, and Function (1995), p. 67-78, editors Soll
and RajBhandary, American Society of Microbiology,
Washington, D.C.). A PCR fragment containing this sequence
was generated from E. coli genomic DNA. The amplification
primer on the 5' side of the gene contained a T7 RNA
polymerise promoter. Thus, the resulting 396 by DNA
amplification product included a T7 promoter immediately 5'
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of the Rnase P RNA gene sequence. This DNA was used as a
template in in vitro transcription reactions using T7
polymerase to generate fluorescently-labeled Rnase P RNA,
as described above. The in vitro transcript produced from
this template was identical in sequence to the published
sequence of E. coli Rnase P RNA.
Example 2. Separation of E. coli Rnase P RNA from 16S
Hha RNA and 16S Alu (see Figures 2A, 2B, 2C
and 2D)
In this example, the ligand is an nucleic acid 5'-CCA
TCG GCG GTT TGC TCT CTG TTG-3' (SEQ ID N0: 4) covalently
immobilized in an standard polyacrylamide gel using the
methods of U.S. patent applications Serial Nos. 08/812,105
and 08/971,845. This ligand is complementary to a sequence
contained within E. coli Rnase P RNA. The ligand was
attached via its 5' terminus, and was present in the gel at
a concentration of 10 mM (strands). The gel contained 5%
polyacrylamide (29:1, monomer:bisacrylamide), 45 mM
Tris-Borate pH 8.3, and 2 mM EDTA (0.5XTBE buffer). The
gel was run in a temperature-controlled minigel apparatus
(Penguin vertical gel box, Owl Scientific, Woburn, MA).
Gel temperature was controlled by pumped water from
external recirculating water baths. For all
electrophoresis steps, the applied field was 200 volts.
The left lane was loaded with fluorescein-labeled E.
coli Rnase P RNA. The right lane was loaded with a mixture
of all three fluorescein-labeled transcripts, Rnase P, 16S
Hha, and 16S Alu.
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The Figure 2A shows the pattern of fluorescent
products after electrophoresis down through the gel for 10
minutes at 54°C, a gel temperature which should disrupt
binding of the gel ligand to the specific analyte, Rnase P
RNA. The gel was imaged directly without removing the
glass plates using a Molecular Dynamics Fluorimager. The
gel was replaced in the gel box and equilibrated at 41°C, a
temperature which should allow hybridization of the nucleic
acid ligand to the Rnase P RNA. The gel ligand is not
complementary to the 16S transcripts. At this temperature,
the samples were electrophoresed upward for 20 minutes.
The field was shut off and the gel temperature was changed
to 59°C. The samples were then electrophoresed down for 5
minutes, and a new gel image of the samples was taken, as
shown in Figure 2B. At this point, only the Rnase P RNA
analytes remain in the gel, demonstrating the efficient
removal of the 16S transcripts during the upward
electrophoresis step. The gel was returned to the
apparatus and electrophoresed up at 41°C for 10 minutes and
down at 59°C for 10 minutes. Another gel image was taken
as shown in Figure 2C. The gel was then electrophoresed
down at 59°C for 25 minutes, and a final image was obtained
as shown in Figure 2D. The Rnase P RNA samples migrate
progressively down the gel as shown in Figures 2C and 2D,
demonstrating release of the specific RNA analytes during
the high temperature downward electrophoresis steps.
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Example 3. Separation of E. coli Rnase P RNA from 16S
Hha RNA, 16S Alu RNA and total unlabeled RNA
from E. coli (see Figures 3A and 3B)
The ligand-containing gel was prepared as in Example
2. Lane 1 contained E. coli Rnase P RNA; lane 2, 16S Alu
transcript; lane 3, 16S Hha and 16S Alu; lane 4, 16S Hha
and Rnase P RNA; lane 5, 6.3 mg total unlabeled RNA from E.
coli; lane 6, Rnase P RNA, 16S Hha, 16S Alu, and 6.3 mg
total unlabeled RNA from E. coli. The gel was equilibrated
to 50°C, a temperature which should disrupt hybridization
of ligand to the specific analyte, Rnase P RNA, and samples
were electrophoresed down for 10 minutes. An image of the
gel after step 1 is seen in Figure 3A. The gel was
equilibrated at 41°C and electrophoresed up for 40 minutes.
At this temperature, the Rnase P RNA can hybridize with the
gel ligand. An image of the gel after step 2, shown in
Figure 3B, demonstrates the complete removal of the 16S Hha
and Alu transcripts from the gel. The specific retention
of Rnase P RNA analyte in the gel was not affected by an
excess of heterogeneous unlabeled RNA in lane 6, further
demonstrating the high specificity of analyte capture.
EQUIVALENTS
While this invention has been particularly shown and
described with references to preferred embodiments thereof,
it will be understood by those skilled in the art that
various changes in form and details may be made therein
without departing from the spirit and scope of the
invention as defined by the appended claims. Those skilled
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in the art will recognize or be able to ascertain using no
more than routine experimentation, many equivalents to the
specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
