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Description  |
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1. FIELD OF THE INVENTION
The present invention relates to a probe composition, and to methods of
using the composition for detecting selected sequences in a target
polynucleotide.
2. REFERENCES
Applied Biosystems, DNA Sequencer User Bulletin, #11, "Synthesis of
Fluorescent Dye-Labeled Oligonucleotides for Use as Primers in
Fluorescence-Based DNA Sequencing (1989).
Blake, et al., Biochemistry, 24:6132 (1985a)
Blake, et al., Biochemistry, 24:6139 (1985b).
Caruthers et al., J. Am Chem Soc, 113 (6324) (1991).
Cohen, A. S., et al., Anal Chem, 59 (7):1021 (1987).
Connell, C., et al., Biotechniques, 5 (342) (1987).
Cload, S. T., et al., J Am Chem Soc, 113: 6324 (1991).
Duck, P., et al., Biotechniques, 9:142 (1989).
Froehler, et al., Nucleic Acids Res, 16:4831 (1988)
Hermans, J. J., J Polymer Sci, 18 (257) (1953).
Holland, et. al., Proc Nat Acad Sci, USA, 88:7276 (1991).
Kornberg, A., et al., "DNA Replication", pp 46-47, W. H. Freeman and Co.,
New York (1992).
Landegren, U., et al., Science, 241:1077 (1988).
Miller, P. S., et al, Biochemistry, 18:5134 (1979).
Miller, P. S., et al., J Biol Chem, 255:6959 (1980).
Miller, P. S., et al., Bioconjugate Chem, 1 (187) (1990).
Mullis, K., et al., U.S. Pat. No. 4,683,202 (1987).
Murakami, et al., Biochemistry, 24:4041 (1985).
Olivera, B. M., et al., Biopolymers, 2 (245) (1964).
Saiki, R. K., et al., Science, 230:1350 (1985).
Sterchak, E. P., et al., Organic Chem, 52:4202 (1987).
Terabe, S., et al., et al., Anal Chem, 57 (4):834 (1985).
Towns, J. K., et al, Anal Chem, 63:1126 (1991).
Whiteley, N. M., et al., U.S. Pat. No. 4,883,750 (1989).
Winn-Deen, E., et al., Clin Chem, 37:1522 (1991).
Wu, D. Y., et al., Genomics, 4:560 (1989).
3. BACKGROUND OF THE INVENTION
A variety of DNA hybridization techniques are available for detecting the
presence of one or more selected polynucleotide sequences in a sample
containing a large number of sequence regions. In a simple method, which
relies on fragment capture and labeling, a fragment containing a seleated
sequence is captured by hybridization to an immobilized probe. The
captured fragment can be labeled by hybridization to a second probe which
contains a detectable reporter moiety.
Another widely used method is Southern blotting. In this method, a mixture
of DNA fragments in a sample are fractionated by gel electrophqresis, then
fixed on nitrocellulose filter. By reacting the filter with one or more
labeled probes under hybridization conditions, the presence of bands
containing the probe sequence can be identified. The method is especially
useful for identifying fragments in a restriction-enzyme DNA digest which
contain a given probe sequence, and for analyzing restriction-fragment
length polymorphisms (RFLPs).
Another approach to detecting the presence of a given sequence or sequences
in a polynucleotide sample involves selective amplification of the
sequence(s) by polymerase chain reaction (Mullis, Saiki). In this method,
primers complementary to opposite end portions of the selected sequence(s)
are used to promote, in conjunction with thermal cycling, successive
rounds of primer-initiated replication. The amplified sequence may be
readily identified by a variety of techniques. This approach is
particularly useful for detecting the presence of low-copy sequences in a
polynucleotide-containing sample, e.g., for detecting pathogen sequences
in a body-fluid sample.
More recently, methods of identifying known target sequences by probe
ligation methods have been reported (Wu, Whiteley, Lundegren, Winn-Deen).
In one approach, known as oligonucleotide ligation assay (OLA), two probes
or probe elements which span a target region of interest are hybridized
with the target region. Where the probe elements match (basepair with)
adjacent target bases at the confronting ends of the probe elements, the
two elements can be joined by ligation, e.g., by treatment with ligase.
The ligated probe element is then assayed, evidencing the presence of the
target sequence.
In a modification of this approach, the ligated probe elements act as a
template for a pair of complementary probe elements. With continued cycles
of denaturation, reannealing and ligation in the presence of the two
complementary pairs of probe elements, the target sequence is amplified
geometrically i.e., exponentially, allowing very small amounts of target
sequence to be detected and/or amplified. This approach is also referred
to as Ligase Chain Reaction (LCR).
There is a growing need, e.g., in the field of genetic screening, for
methods useful in detecting the presence or absence of each of a large
number of sequences in a target polynucleotide. For example, as many as
150 different mutations have been associated with cystic fibrosis. In
screening for genetic predisposition to this disease, it is optimal to
test all of the possible different gene sequence mutations in the
subject's genomic DNA, in order to make a positive identification of a
"cystic fibrosis". Ideally, one would like to test for the presence or
absence of all of the possible mutation sites in a single assay.
These prior-art methods described above are not readily adaptable for use
in detecting multiple selected sequences in a convenient, automated
single-assay format. It is therefore desirable to provide a rapid,
single-assay format for detecting the presence or absence of multiple
selected sequences in a polynucleotide sample.
4. SUMMARY OF THE INVENTION
The present invention includes, in one aspect, a method of detecting one or
more of a plurality of different sequences in a target polynucleotide. In
practicing the method, there is added to the target polynucleotide, a
plurality of sequence-specific probes, each characterized by (a) a binding
polymer having a probe-specific sequence of subunits designed for
base-specific binding of the polymer to one of the target sequences, under
selected binding conditions, and (b) attached to the binding polymer, a
polymer chain having a different ratio of charge/translational frictional
drag from that of the binding polymer.
The probes are reacted with the target polynucleotide under conditions
favoring binding of the probes in a base-specific manner to the target
polynucleotide. The probes are then treated to selectively modify those
probes which are bound to the target polynucleotide in a sequence-specific
manner, forming modified, labeled probes characterized by (a) a
distinctive ratio of charge/translational frictional drag, and (b) a
detectable reporter label.
The modified, labeled probes are fractionated by electrophoresis in a
non-sieving matrix. The presence of selected sequence(s) in the target
polynucleotide is detected according to the observed electrophoretic
migration rates of the labeled probes.
The polymer chain may be a substantially uncharged, water-soluble chain,
such as a chain composed of polyethylene oxide (PEO) units or a
polypeptide chain, where the chains attached to different-sequence binding
polymers have different numbers of polymer units. Electrophoresis is
preferably performed under conditions of efficient heat dissipation from
the non-sieving medium, such as in a capillary tube.
In one general method, each probe includes first and second probe elements
having first and second sequence-specific oligonucleotides which, when
bound in a sequence specific manner to a selected single-stranded target
sequence, have (or can be modified to have) confronting end subunits which
can basepair to adjacent bases in the target polynucleotide sequence.
After hybridizing the oligonucleotides to the target polynucleotide, the
target-bound oligonucleotides are ligated, to join those hybridized
oligonucleotides whose confronting end subunits are base-paired with
adjacent target bases. In each pair of probe elements, one of the probe
elements contains the probe-specific polymer chain, and the other element
preferably includes a detectable reporter.
In a second general embodiment, each probe includes first and second primer
elements having first and second sequence-specific oligonucleotide primers
effective to hybridize with opposite end regions of complementary strands
of a selected target polynucleotide segment. The first probe element
contains the probe-specific polymer chain. The primer elements are reacted
with the target polynucleotide in a series of primer-initiated
polymerization cycles which are effective to amplify the target sequence
of interest.
The amplification reaction may be carried out in the presence of
reporter-labeled nucleoside triphosphates, for purposes of reporter
labeling the amplified sequences. Alternatively, the amplified target
sequences may be labeled, in single-stranded form, by hybridization with
one or more reporter-labeled, sequence-specific probes, or in
double-stranded form by covalent or non-covalent attachment of a reporter,
such as ethidium bromide.
In a third general embodiment, bound oligonucleotide probes are reacted
with reporter-labeled nucleoside triphosphate molecules, in the presence
of a DNA polymerase, to attach reporter groups to the 3' end of the
probes.
In a fourth general embodiment, each probe includes binding polymer which
is modified by enzymatic cleavage when bound to a target sequence. The
cleavage reaction may remove a portion of the binding polymer, to modify
the probes's ratio of charge/translational frictional drag, or may
separate a reporter label carried at one end of the binding polymer from a
polymer chain carried at the other end of the binding polymer, to modify
the charge/translational frictional drag of the binding polymer carrying
the reporter label.
In a fifth general embodiment, each sequence-specific probe includes a
binding polymer and an attached reporter label, and the polymer chain
associated with each different-sequence probe imparts to that probe, a
distinctive ratio of charge/translational frictional drag. The treating
step includes immobilizing the target polynucleotide, washing the
immobilized target polynucleotide to remove probes not bound to the target
polynucleotide in a sequence-specific manner, and denaturing the target
polynucleotide to release probes bound in a sequence-specific manner.
Also forming part of the invention is a probe composition for use in
detecting one or more of a plurality of different sequences in a target
polynucleotide. The composition includes a plurality of sequence-specific
probes, each characterized by (a) a binding polymer having a
probe-specific sequence of subunits designed for base-specific binding of
the polymer to one of the target sequences, under selected binding
conditions, and (b) attached to the binding polymer, a polymer chain
having a ratio of charge/translational frictional drag which is different
from that of the binding polymer.
In one embodiment, each sequence specific probe further includes a second
binding polymer, where the first-mentioned and second binding polymers in
a sequence-specific probe are effective to bind in a base-specific manner
to adjacent and contiguous regions of a selected target sequence, allowing
ligation of the two binding polymers when bound to the target sequence in
a sequence-specific manner. The second binding polymer preferably includes
a detectable label, and the polymer chain attached to the first binding
polymer imparts to each ligated probe pair, a distinctive combined ratio
of charge/translational frictional drag.
In another embodiment, each sequence specific probe in the composition
further includes a second binding polymer, where the first-mentioned and
second binding polymers in a sequence-specific probe are effective to bind
in a base-specific manner to opposite end regions of opposite strands of a
selected duplex target sequence, allowing primer initiated polymerization
of the target region in each strand. The second binding polymer preferably
includes a detectable label, and the polymer chain attached to the first
binding polymer imparts to each ligated probe pair, a distinctive combined
ratio of charge/translational frictional drag.
In another embodiment, each sequence-specific probe includes a binding
polymer, a polymer chain attached to the binding polymer, and a reporter
attached to the binding polymer.
These and other objects and features of the invention will become more
fully apparent when the following detailed description of the invention is
read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1D illustrate three general types of probes and probe elements
used in practicing various embodiments of the method of the invention;
FIG. 2 illustrates methods of synthesis of polyethylene oxide polymer
chains having a selected number of hexapolyethylene oxide (HEO) units;
FIG. 3 illustrates methods of synthesis of polyethylene glycol polymer
chains in which HEO units are linked by bisurethane tolyl linkages;
FIGS. 4A and 4B illustrate coupling reactions for coupling the polymer
chains of FIGS. 2 and 3 to the 5' end of a polynucleotide, respectively;
FIG. 5 shows the reaction steps for adding HEO units successively to an
oligonucleotide through phosphodiester linkages, and subsequent
fluorescent tagging;
FIG. 6 is an electropherogram, on capillary electrophoresis in a
non-sieving medium, of a 24 base oligonucleotide before (peak 1) and after
derivatization with 1 (peak 2), 2 (peak 3), and 4 (peak 4) units of a
hexaethylene oxide (HEO) unit;
FIGS. 7A-7D illustrate a probe-ligation method of identifying target
sequences, in accordance with a first general embodiment of the method of
the invention;
FIG. 8 illustrates an idealized electrophoretic pattern observed in the
FIG. 7 method, where a target polynucleotide contains mutations in two
different target regions;
FIG. 9 is an electropherogram, on capillary electrophoresis, in a
non-sieving medium, of labeled probes having polypeptide polymer chains,
and formed by ligation of adjacent probes on a target molecule;
FIGS. 10A-10C illustrate a method of detecting target sequences by ligation
of base-matched probes by ligase chain reaction (LCR) in accordance with
the first general embodiment of the invention;
FIG. 11 is an electropherogram, on capillary electrophoresis in a
non-sieving matrix, of labeled probes having polyethylene oxide polymer
chains, and formed by LCR reaction;
FIGS. 12A-12B illustrate the steps in a second general embodiment of the
invention, using primer-initiated amplification to produce double-stranded
labeled probes;
FIGS. 13A and 13B illustrate an alternative method for labeling amplified
target sequences formed in the FIG. 12 method;
FIGS. 14A and 14B illustrate steps in a third general embodiment of the
invention, using reporter-labeled nucleotide addition to the target-bound
probes to form labeled probe species;
FIGS. 15A and 15B illustrate a method for labeling target duplex fragments
with polymer chains, for purposes of identifying fragments containing
selected sequences, in accordance with the second general embodiment of
the method of the invention;
FIGS. 16A-16C illustrate an alternative method for modifying probes in a
sequence specific manner to contain both polymer chains and reporter
labels, in accordance with the first general embodiment of the method of
the invention;
FIGS. 17A and 17B illustrate a method for identifying target sequences by
selective probe cleavage, in accordance with a fourth general embodiment
of the invention;
FIGS. 18A-18B illustrate an alternative probe-ligation method, in
accordance with the first general embodiment of the invention;
FIGS. 19A and 19B illustrate a method for modifying labeled probes by
polymerase cleavage reaction, in accordance with the fourth general
embodiment of the invention; and
FIGS. 20A-20C illustrate steps in a probe capture method of identifying
target sequences, in accordance with a fifth general embodiment of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
"A target polynucleotide" may include one or more nucleic acid molecules,
including linear or circularized single-stranded or double-stranded RNA or
DNA molecules.
"Target nucleic acid sequence" means a contiguous sequence of nucleotides
in the target polynucleotide. A "plurality" of such sequences includes two
or more nucleic acid sequences differing in base sequence at one or more
nucleotide positions.
"Sequence-specific binding polymer" means a polymer effective to bind to
one target nucleic acid or sequence subset with base-sequence specificity,
and which has a substantially lower binding affinity, under selected
hybridization conditions, to any other target sequence or sequence subset
in a given plurality of sequences in the test sample.
The "charge" of a polymer is the total net electrostatic charge of the
polymer at a given pH;
The "translational frictional drag" of a polymer is a measure of the
polymer's frictional drag as it moves electrophoretically through a
defined, non-sieving liquid medium.
"Non-sieving matrix" means a liquid medium which is substantially free of a
mesh or network or matrix of interconnected polymer molecules.
A "distinctive ratio of charge/translational frictional drag" of a probe is
evidenced by a distinctive, i.e., unique, electrophoretic mobility of the
probe in a non-sieving medium.
"Capillary electrophoresis" means electrophoresis in a capillary tube or in
a capillary plate, where the diameter of separation column or thickness of
the separation plate is between about 25-500 microns, allowing efficient
heat dissipation throughout the separation medium, with consequently low
thermal convection within the medium.
A "labeled probe" refers to a probe which is composed of a binding polymer
effective to bind in a sequence-specific manner to a selected target
sequence, a polymer chain which imparts to the binding polymer, a
distinctive ratio of charge/translational frictional drag, and a
detectable reporter or tag.
A "reporter" or "label" or "reporter label" refers to a fluorophore,
chromophore, radioisotope, or spin label which allows direct detection of
a labeled probe by a suitable detector, or a ligand, such as an antigen,
or biotin, which can bind specifically and with high affinity to a
detectable anti-ligand, such as a reporter-labeled antibody or avidin.
II. probe Composition
This section describes several embodiments of probes designed for use in
the present invention. In the typical case, the probe is part of a probe
composition which contains a plurality of probes used for detecting one or
more of a plurality of target sequences, according to methods described in
Section III. The probes described with reference to FIG. 1B and 1C are
representative of probes or probe elements which make up probe
compositions in accordance with the present invention.
A. probe Structure
FIG. 1 shows a probe 20 which is one of a plurality of probes used in one
embodiment of the method of the invention. As will be seen below, a probe
composition containing a probe like probe 20 is designed for use in a
"probe-extension" method of identifying target sequences, such as the
sequence in region 24 of a target polynucleotide, indicated by dashed line
at 26 in FIG. 1A, or in a "probe-capture" method for identifying such
target sequences. Both methods are discussed in Section IV below.
Probe 20 includes an oligonucleotide binding polymer 22 which preferably
includes at least 10-20 bases, for requisite basepair specificity, and has
a base sequence which is complementary to region 24 in target
polynucleotide 26, with such in single-stranded form. Other probes in the
composition have sequence specificities for other target regions of known
sequence in the target polynucleotide. In a preferred embodiment, the
binding polymers of the different-sequence probes all have about the same
length, allowing hybridization of the different probes to the target
polynucleotide with substantially the same hybridization reaction kinetics
and thermodynamics (T.sub.m).
Other binding polymers which are analogs of polynucleotides, such as
deoxynucleotides with thiophosphodiester linkages, and which are capable
of base-specific binding to single-stranded or double-stranded target
polynucleotides are also contemplated. Polynucleotide analogs containing
uncharged, but stereoisomeric methylphosphonate linkages between the
deoxyribonucleoside subunits have been reported (Miller, 1979, 1980, 1990,
Murakami, Blake, 1985a, 1985b). A variety of analogous uncharged
phosphoramidate-linked oligonucleotide analogs have also been reported
(Froehler). Also, deoxyribonucleoside analogs having achiral and uncharged
intersubunit linkages (Sterchak) and uncharged morpholino-based polymers
having achiral intersubunit linkages have been reported (U.S. Pat. No.
5,034,506). Such binding polymers may be designed for sequence specific
binding to a single-stranded target molecule through Watson-Crick base
pairing, or sequence-specific binding to a double-stranded target
polynucleotide through Hoogstein binding sites in-the major groove of
duplex nucleic acid (Kornberg).
The binding polymer in the probe has a given ratio of charge/translational
frictional drag, as defined above, and this ratio may be substantially the
same for all of the different-sequence binding polymers of the plurality
of probes making up the probe composition. This is evidenced by the
similar migration rates of oligonucleotides having different sizes
(numbers of subunits) and sequences by electrophoresis in a non sieving
medium.
The oligonucleotide binding polymer in probe 20 is derivatized, at its 5'
end, with a polymer chain 27 composed of N subunits 28. The units may be
the subunits of the polymer or may be groups of subunits. Exemplary
polymer chains are formed of polyethylene oxide, polyglycolic acid,
polylactic acid, polypeptide, oligosaccharide, polyurethane, polyamids,
polysulfonamide, polysulfoxide, and block copolymers thereof, including
polymers composed of units of multiple subunits linked by charged or
uncharged linking groups.
According to an important feature of the invention, the polymer chain has a
ratio of charge/translational frictional drag which is different from that
of the binding polymer. In the method of the invention, detailed in
Section IV below, the probes are treated to selectively modify those
probes bound in a sequence-specific manner to a target sequence, to
produce modified, labeled probes having a distinct ratio of
charge/translational frictional coefficient, as evidenced by a distinctive
electrophoretic mobility in a non-sieving matrix, as discussed in Section
III below. As will be discussed below, the distinctive ratio of
charge/translational frictional drag is typically achieved by differences
in the lengths (number of subunits) of the polymer chain. However,
differences in polymer chain charge are also contemplated, as are
differences in binding-polymer length.
More generally, the polymers forming the polymer chain may be homopolymers,
random copolymers, or block copolymers, and the polymer may have a linear,
comb, branched, or dendritic architecture. In addition, although the
invention is described herein with respect to a single polymer chain
attached to an associated binding polymer at a single point, the invention
also contemplates binding polymers which are derivatized by more than one
polymer chain element, where the elements collectively form the polymer
chain.
Preferred polymer chains are those which are hydrophilic, or at least
sufficiently hydrophilic when bound to the oligonucleotide binding polymer
to ensure that the probe is readily soluble in aqueous medium. The polymer
chain should also not affect the hybridization reaction. Where the binding
polymers are highly charged, as in the case of oligonucleotides, the
polymer chains are preferably uncharged or have a charge/subunit density
which is substantially less than that of the binding polymer.
Methods of synthesizing selected-length polymer chains, either separately
or as part of a single-probe solid-phase synthetic method, are described
below, along with preferred properties of the polymer chains.
In one preferred embodiment, described below, the polymer chain is formed
of hexaethylene oxide (HEO) units, where the HEO units are joined
end-to-end to form an unbroken chain of ethylene oxide subunits, as
illustrated in FIG. 2, or are joined by charged (FIG. 5) or uncharged
(FIG. 3) linkages, as described below. Other embodiments exemplified below
include a chain composed of N 12 mer PEO units, and a chain composed of N
tetrapeptide units.
B. Probe Compositions
This section describes three additional probes or probe-element pairs which
are useful in specific embodiments of the method of the invention and
which themselves, either as single probes or as probe sets, form
compositions in accordance with the invention.
FIG. 1B illustrates a probe 25 which has a sequence-specific
oligonucleotide binding polymer 21 designed for sequence-specific, i.e.,
base-specific binding to a region of a target polynucleotide 23. By this
is meant the binding polymer contains a sequence of subunits effective to
form a stable duplex or triplex hybrid with the selected single-stranded
or double-stranded target sequence, respectively, under defined
hybridization conditions. As will be seen with reference to FIG. 17 below,
the binding polymer may contain both DNA and RNA segments. Attached to the
binding polymer, at its 5' end, is a polymer chain 31 composed of N units
33, which imparts to the binding polymer a distinctive ratio of
charge/translational frictional drag, as described above. The 3' end of
the binding polymer is derivatized with a reporter or tag 39. In one
aspect, the invention includes a composition which includes a plurality of
such probes, each with a different-sequence binding polymer targeted
against different target regions of interest, and each with a distinctive
ratio of charge/translational frictional drag imparted by the associated
polymer chain.
FIG. 1C illustrates a probe 32 which consists of first and second probe
elements 34, 36, is designed particularly for detecting selected sequences
in each of one or more regions, such as region 38, of a target
polynucleotide, indicated by dashed line 40.
In the embodiment illustrated, the sequences of interest may involve
mutations, for example, point mutations, or addition or deletion type
mutations involving one or a small number of bases. In a typical example,
the expected site of mutation is near the midpoint of the known-sequence
target region, and divides that region into two subregions. In the example
shown, the mutation is a point mutation, and the expected site of the
mutation is at one of the two adjacent bases T-G, with the T base defining
the 5' end of a subregion 38a, and the adjacent G base, defining the 3'
end of adjacent subregion 38b. As will be seen below, the probe elements
are also useful for detecting a variety of other types of target
sequences, e.g., sequences related to pathogens or specific genomic gene
sequences.
Probe element 32, which is representative of the first probe elements in
the probe composition, is composed of an oligonucleotide binding polymer
element 42 which preferably includes at least 10-20 bases, for requisite
basepair specificity, and has a base sequence which is complementary to a
subregion 38a in the target polynucleotide. In particular, the 3' end
nucleotide bases are selected for base pairing to the 5' end nucleotide
bases of the corresponding subregion, e.g., the A:T matching indicated.
The oligonucleotide in the first probe element is derivatized, at its 5'
end, with a polymer chain 44 composed of N preferably repeating units 45,
substantially as described with respect to chain 27 formed from units 28.
As described with respect to probe 20, the polymer chain in the first
probe element imparts a ratio of charge/translational frictional drag
which is distinctive for each sequence-specific probe element in the
composition.
Second probe element 36, which is also representative of the second probe
elements in | | |
