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Claims  |
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What is claimed is:
1. A reagent composition which comprises an aqueous carrier, a primer
nucleic acid molecule, a template nucleic acid of interest hybridized
thereto, and an admixture of at least two different terminators of a
nucleic acid template-dependent, primer extension reaction, each of the
terminators being capable of specifically terminating an extension
reaction of said primer hybridized to said template nucleic acid molecule
of interest in a manner strictly dependent on the identity of the unpaired
nucleotide base in said template nucleic acid molecule of interest
immediately adjacent to, and downstream of, the 3' end of said primer, and
at least one of the terminators being labeled with a detectable marker,
and wherein said reagent composition lacks dATP, dCTP, dGTP, and dTTP.
2. A reagent of claim 1, wherein the reagent comprises four different
terminators.
3. A reagent of claim 2, wherein two of the terminators are labeled, each
with a different detectable marker.
4. A reagent of claim 2, wherein three of the terminators are labeled, each
with a different detectable marker.
5. A reagent of claim 2, wherein the four terminators are labeled, each
with a different detectable marker.
6. A reagent of any of claims 1-5, wherein the terminator(s) comprise(s) a
nucleotide or nucleotide analog.
7. A reagent of claim 6, wherein the terminator(s) comprise(s)
dideoxynucleotides.
8. A reagent of claim 6, wherein the terminator(s) comprise(s) arabinoside
triphosphates.
9. A reagent of claim 7, wherein the terminator(s) comprise(s) one or more
of ddATP, ddCTP, ddGTP or ddTTP.
10. A reagent of any of claims 1-5, wherein each of the different
detectable markers is an isotopically labeled moiety, a chromophore, a
fluorophore, a protein moiety, or a moiety to which an isotopically
labeled moiety, a chromophore, a fluorophore, or a protein moiety can be
attached.
11. A reagent of claim 10, wherein each of the different detectable markers
is a different fluorophore.
12. A reagent of any of claims 1-5, wherein the reagent further comprises
pyrophosphatase.
13. A method of determining the identity of a nucleotide base at a specific
position in a nucleic acid of interest which comprises:
(a) treating a sample containing the nucleic acid of interest, if such
nucleic acid is double-stranded, so as to obtain unpaired nucleotide bases
spanning the specific position, or directly employing step (b) if the
nucleic acid of interest is single-stranded;
(b) contacting the sample from step (a), with an oligonucleotide primer,
said primer being immobilized to a solid support wherein said primer is
fully complementary to and hybridizes specifically to a stretch of
nucleotide bases present in the nucleic acid of interest, immediately
adjacent to the nucleotide base to be identified, under high stringency
hybridization conditions, so as to form a duplex between the immobilized
primer and the nucleic acid of interest such that the nucleotide base to
be identified is the first unpaired base in the template immediately
downstream of the 3' end of the primer in said duplex;
(c) contacting the duplex from step (b) in the absence of dATP, dCTP, dGTP,
or dTTP, with at least two different terminators of a nucleic acid
template-dependent, primer extension reaction capable of specifically
terminating the extension reaction in a manner strictly dependent on the
identity of the unpaired nucleotide base in the template nucleic acid of
interest immediately downstream of the 3' end of the immobilized primer;
wherein one of said terminators is complementary to said nucleotide base
to be identified and wherein at least one of said terminators is labeled
with a detectable marker, wherein if more than one terminator is labeled,
a different label is used to label each such labeled terminator; wherein
said contacting is under conditions sufficient to permit base pairing of
said complementary terminator with the nucleotide base to be identified
and occurrence of a template-dependent primer extension reaction
sufficient to incorporate said complementary terminator onto the 3' end of
the primer to thereby extend said 3' end of said primer by one terminator;
(d) determining the presence and identity of the nucleotide base at the
specific position in the nucleic acid of interest by detecting the
detectable marker of said incorporated terminator while said terminator is
incorporated at the 3' end of the extended primer, and wherein said
detection is conducted in the absence of non-terminator nucleotides.
14. A method of determining the presence or absence of a desired
polynucleotide having a particular nucleotide sequence in a sample of
nucleic acids which comprises:
(a) treating a sample of nucleic acids, if such sample of nucleic acids
contains double-stranded nucleic acids, so as to obtain single-stranded
nucleic acids, or directly employing step (b) if the sample of nucleic
acids contains only single-stranded nucleic acids;
(b) contacting the sample from step (a), with an oligonucleotide primer,
said primer being immobilized to a solid support; wherein said primer is
fully complementary to and hybridizes specifically to a stretch of
nucleotide bases of the particular nucleotide sequence, if the desired
polynucleotide having said particular nucleotide sequence is present, said
contacting being under high stringency hybridization conditions, so as to
form a duplex between the immobilized primer and the particular nucleotide
sequence;
(c) contacting the duplex, if any, from step (b) in the absence of dATP,
dCTP, dGTP or dTTP, with at least two different terminators of a nucleic
acid template-dependent, primer extension reaction capable of specifically
terminating the extension reaction in a manner strictly dependent on the
identity of the unpaired nucleotide base in the template immediately
downstream of the 3' end of the immobilized primer; wherein one of said
terminators is complementary to said unpaired nucleotide base and wherein
at least one of said terminators is labeled with a detectable marker,
wherein if more than one terminator is labeled, a different label is used
to label each such labeled terminator; wherein said contacting is under
conditions sufficient to permit base pairing of said complementary
terminator with said unpaired nucleotide base immediately downstream of
the 3' end of said immobilized hybridized primer and occurrence of a
template-dependent, primer extension reaction sufficient to incorporate
said complementary terminator onto the 3' end of the immobilized primer to
thereby extend said 3' end of said primer by one terminator; and
(d) determining the presence of the target nucleic acid molecule by
detecting the detectable marker of said incorporated terminator while said
terminator is incorporated at the 3' end of the extended primer from step
(c) and wherein said detection is conducted in the absence of
non-terminator nucleotides.
15. A method of determining the identity of a nucleotide base at a specific
position in a nucleic acid of interest which comprises:
(a) (1) incubating a sample containing the nucleic acid of interest with at
least two oligonucleotide primers, and a polymerase, said primers being
sufficient to mediate a polymerase chain reaction amplification of said
nucleic acid of interest, wherein said incubation is conducted under
conditions sufficient to permit said amplification to occur;
(2) treating a sample containing said amplified nucleic acid of interest,
if such nucleic acid is double-stranded, so as to obtain unpaired
nucleotide bases spanning the specific position, or directly employing
step (b) if the nucleic acid of interest is single-stranded;
(b) contacting the sample from step (a2), under hybridizing conditions,
with an oligonucleotide primer, said primer being immobilized to a solid
support wherein said primer is fully complementary to and hybridizes
specifically to a stretch of nucleotide bases present in the nucleic acid
of interest immediately adjacent to the nucleotide base to be identified,
so as to form a duplex between the immobilized primer and the nucleic acid
of interest such that the nucleotide base to be identified is the first
unpaired base in the template immediately downstream of the 3' end of the
primer in said duplex;
(c) contacting the duplex from step (b), in the absence of dATP, dCTP,
dGTP, or dTTP, with at least two different terminators of a nucleic acid
template-dependent, primer extension reaction capable of specifically
terminating the extension reaction in a manner strictly dependent upon the
identity of the unpaired nucleotide base in the template nucleic acid of
interest immediately downstream of the 3' end of the immobilized primer;
wherein one of said terminators is complementary to said nucleotide base
to be identified and wherein at least one of said terminators is labeled
with a detectable marker, wherein if more than one terminator is labeled,
a different label is used to label each such labeled terminator; wherein
said contacting is under conditions sufficient to permit base pairing of
said complementary terminator with the nucleotide base immediately
downstream of the 3' end of said hybridized primer and occurrence of a
template-dependent primer extension reaction sufficient to incorporate
said complementary terminator onto the 3' end of the primer to thereby
extend said 3' end of said primer by one terminator;
(d) determining the presence and identity of the nucleotide base at the
specific position in the nucleic acid of interest by detecting the
detectable marker of said incorporated terminator while said terminator is
incorporated at the 3' end of the extended primer, and wherein said
detection is conducted in the absence of non-terminator nucleotides.
16. A method of determining the presence or absence of a desired
polynucleotide having a particular nucleotide sequence in a sample of
nucleic acids which comprises:
(a) (1) incubating a sample of nucleic acids with at least two
oligonucleotide primers, and a polymerase, said primers being sufficient
to mediate a polymerase chain reaction amplification of said nucleic acid
of interest, if said nucleic acid of interest is present in said sample,
wherein said incubation is conducted under conditions sufficient to permit
said amplification to occur;
(2) treating a sample containing said amplified nucleic acid of interest,
if such nucleic acid is double-stranded, so as to obtain unpaired
nucleotide bases spanning the specific position, or directly employing
step (b) if the nucleic acid of interest is single-stranded;
(b) contacting the sample from step (a2), under hybridizing conditions,
with an oligonucleotide primer, said primer being immobilized to a solid
support; wherein said primer is fully complementary to and hybridizes
specifically to a stretch of nucleotide bases of the particular nucleotide
sequence, if the desired polynucleotide having said particular nucleotide
sequence is present, so as to form a duplex between the immobilized primer
and the particular nucleotide sequence;
(c) contacting the duplex, if any, from step (b), in the absence of dATP,
dCTP, dGTP or dTTP, with at least two different terminators of a nucleic
acid template-dependent, primer extension reaction capable of specifically
terminating the extension reaction in a manner strictly dependent upon the
identity of the unpaired nucleotide base in the template immediately
downstream of the 3' end of the immobilized primer; wherein one of said
terminators is complementary to said unpaired nucleotide base and wherein
at least one of said terminators is labeled with a detectable marker,
wherein if more than one terminator is labeled, a different label is used
to label each such labeled terminator; wherein said contacting is under
conditions sufficient to permit base pairing of said complementary
terminator with said unpaired nucleotide base immediately downstream of
the 3' end of said immobilized hybridized primer and occurrence of a
template-dependent, primer extension reaction sufficient to incorporate
said complementary terminator onto the 3' end of the immobilized primer to
thereby extend said 3' end of said primer by one terminator; and
(d) determining the presence of the target nucleic acid molecule by
detecting the detectable marker of said incorporated terminator while said
terminator is incorporated at the 3' end of the extended primer from step
(c) and wherein said detection is conducted in the absence of
non-terminator nucleotides.
17. A method of typing a sample containing nucleic acids which comprises
identifying the nucleotide base or bases present at each of one or more
specific positions, each such nucleotide base being identified using the
method of claim 13 or 15, and each such specific position being determined
using a different primer.
18. A method of claim 17, wherein the identity of each nucleotide base or
bases at each position is determined individually or wherein the
identities of the nucleotide bases at different positions are determined
simultaneously.
19. A method of typing a sample containing nucleic acids which comprises
determining the presence or absence of one or more particular nucleotide
sequences, the presence or absence of each such nucleotide sequence being
determined by the method of claim 14 or 16.
20. A method for identifying different alleles in a sample containing
nucleic acids which comprises identifying the nucleotide base or bases
present at each of one or more specific positions, each such nucleotide
base being identified by the method of claim 13 or 16.
21. A method for determining the genotype of an organism at one or more
particular genetic loci which comprises:
(a) obtaining from the organism a sample containing genomic DNA; and
(b) identifying the nucleotide base or bases present at each of one or more
specific positions in nucleic acids of interest, each such base or bases
being identified using the method of claim 13 or 15, and thereby
identifying different alleles and thereby, in turn, determining the
genotype of the organism at one or more particular genetic loci.
22. A method of claim 13 or 15, wherein the conditions for the occurrence
of the template-dependent, primer extension reaction in step (c) are
created, in part, by the presence of a suitable template-dependent enzyme.
23. A method of claim 22, wherein the template-dependent enzyme is E. coli
DNA polymerase I or the "Klenow fragment" thereof, T4 DNA polymerase, T7
DNA polymerase ("Sequenase"), T. aquaticus DNA polymerase, a retroviral
reverse transcriptase, or combinations thereof.
24. A method of claim 13 or 15, wherein the nucleic acid of interest is a
deoxyribonucleic acid, a ribonucleic acid, or a copolymer of
deoxyribonucleic acid and ribonucleic acid.
25. A method of claim 13 or 15, wherein the primer is an
oligodeoxyribonucleotide, an oligoribonucleotide, or a copolymer of
deoxyribonucleic acid and ribonucleic acid.
26. A method of claim 13 or 15, wherein the template is a deoxyribonucleic
acid, the primer is an oligodeoxyribonucleotide, oligoribonucleotide, or a
copolymer of deoxyribonucleotides and ribonucleotides, and the
template-dependent enzyme is a DNA polymerase.
27. A method of claim 13 or 15, wherein the template is a ribonucleic acid,
the primer is an oligodeoxyribonucleotide, oligoribonucleotide, or a
copolymer of deoxyribonucleotides and ribonucleotides, and the
template-dependent enzyme is a reverse transcriptase.
28. A method of claim 13 or 15, wherein the template is a deoxyribonucleic
acid, the primer is an oligoribonucleotide, and the enzyme is an RNA
polymerase.
29. A method of claim 13 or 15, wherein the template is a ribonucleic acid,
the primer is an oligoribonucleotide, and the template-dependent enzyme is
an RNA replicase.
30. A method of claim 13 or 15, wherein, prior to the primer extension
reaction in step (c), the template has been capped at its 3' end by the
addition of a terminator to the 3' end of the template, said terminator
being capable of terminating a template-dependent, primer extension
reaction.
31. A method of claim 30, wherein the terminator is a dideoxynucleotide.
32. A method of claim 13 or 15, wherein the nucleic acid of interest has
been synthesized enzymatically in vivo, synthesized enzymatically in
vitro, or synthesized non-enzymatically.
33. A method of claim 13 or 15, wherein the oligonucleotide primer has been
synthesized enzymatically in vivo, synthesized enzymatically in vitro, or
synthesized non-enzymatically.
34. A method of claim 13 or 15, wherein the oligonucleotide primer
comprises one or more moieties that permit affinity separation of the
primer from the unincorporated reagent and/or the nucleic acid of
interest.
35. A method of claim 34, wherein the oligonucleotide primer comprises
biotin which permits affinity separation of the primer from the
unincorporated reagent and/or nucleic acid of interest via binding of the
biotin to streptavidin which is attached to a solid support.
36. A method of claim 13 or 15, wherein the sequence of the oligonucleotide
primer comprises a DNA sequence that permits affinity separation of the
primer from the unincorporated reagent and/or the nucleic acid of interest
via base pairing to a complementary sequence present in a nucleic acid
attached to a solid support.
37. A method of claim 13 or 15, wherein the nucleic acid of interest
comprises one or more moieties that permit affinity separation of the
nucleic acid of interest from the unincorporated reagent and/or the
primer.
38. A method of claim 37, wherein the nucleic acid of interest comprises
biotin which permits affinity separation of the nucleic acid of interest
from the unincorporated reagent and/or the primer via binding of the
biotin to streptavidin which is attached to a solid support.
39. A method of claim 13 or 15, wherein the sequence of the nucleic acid of
interest comprises a DNA sequence that permits affinity separation of the
nucleic acid of interest from the unincorporated reagent and/or the primer
via base pairing to a complementary sequence present in a nucleic acid
attached to a solid support.
40. A method of claim 13 or 15, wherein the oligonucleotide primer is
labeled with a detectable marker.
41. A method of claim 40, wherein the oligonucleotide primer is labeled
with a detectable marker that is different from any detectable marker
present in the reagent or attached to the nucleic acid of interest.
42. A method of claim 13 or 15, wherein the nucleic acid of interest is
labeled with a detectable marker.
43. A method of claim 42, wherein the nucleic acid of interest is labeled
with a detectable marker that is different from any detectable marker
present in the reagent or attached to the primer.
44. A method of claim 13 or 15, wherein the nucleic acid of interest
comprises non-natural nucleotide analogs.
45. A method of claim 44, wherein the non-natural nucleotide analogs
comprise deoxyinosine or 7-deaza-2'-deoxyguanosine.
46. A method of claim 13 or 15, wherein the nucleic acid of interest has
been synthesized by the polymerase chain reaction.
47. A method of claim 13 or 15, wherein the sample comprises genomic DNA
from an organism, RNA transcripts thereof, or cDNA prepared from RNA
transcripts thereof.
48. A method of claim 13 or 15, wherein the sample comprises extragenomic
DNA from an organism, RNA transcripts thereof, or cDNA prepared from RNA
transcripts thereof.
49. A method of claim 13 or 15, wherein the primer is separated from the
nucleic acid of interest after the primer extension reaction in step (c)
by using appropriate denaturing conditions.
50. A method of claim 49, wherein the denaturing conditions comprise heat,
alkali, formamide, urea, glyoxal, enzymes, and combinations thereof.
51. A method of claim 50, wherein the denaturing conditions comprise
treatment with 0.2 N NaOH.
52. A method of claim 47, wherein the organism is a plant, microorganism,
virus, or bird.
53. A method of claim 47, wherein the organism is a vertebrate or
invertebrate.
54. A method of claim 47, wherein the organism is a mammal.
55. A method of claim 54, wherein the mammal is a human being.
56. A method of claim 54, wherein the mammal is a horse, dog, cow, cat,
pig, or sheep. |
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Claims |
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Description |
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BACKGROUND OF THE INVENTION
This invention relates to the field of nucleic acid sequence detection. The
detection of nucleic acid sequences can be used in two general contexts.
First, the detection of nucleic acid sequences can be used to determine
the presence or absence of a particular genetic element. Second, the
detection of nucleic acid sequences can be used to determine the specific
type of a particular genetic element that is present. Variant genetic
elements usually exist. Many techniques have been developed (1) to
determine the presence of specific nucleic acid sequences, and (2) to
compare homologous segments of nucleic acid sequence to determine if the
segments are identical or if they differ at one or more nucleotides.
Practical applications of these techniques include genetic disease
diagnoses, infectious disease diagnoses, forensic techniques, paternity
determinations, and genome mapping.
In general, the detection of nucleic acids in a sample and the subtypes
thereof depends on the technique of specific nucleic acid hybridization in
which the oligonucleotide probe is annealed under conditions of high
stringency to nucleic acids in the sample, and the successfully annealed
probes are subsequently detected (see Spiegelman, S., Scientific American,
Vol. 210, p. 48 (1964)).
The most definitive method for comparing DNA segments is to determine the
complete nucleotide sequence of each segment. Examples of how sequencing
has been used to study mutations in human genes are included in the
publications of Engelke, et al., Proc. Natl. Acad. Sci. U.S.A., 85:544-548
(1988) and Wong, et al., Nature, 330:384-386 (1987). At the present time,
it is not practical to use extensive sequencing to compare more than just
a few DNA segments because the effort required to determine, interpret,
and compare sequence information is time-consuming.
A commonly used screen for DNA polymorphisms arising from DNA sequence
variation consists of digesting DNA with restriction endonucleases and
analyzing the resulting fragments by means of Southern blots, as described
by Botstein, et al., Am. J. Hum. Genet., 32:314-331 (1980) and White, et
al., Sci. Am., 258:40-48 (1988). Mutations that affect the recognition
sequence of the endonuclease will preclude enzymatic cleavage at that
site, thereby altering the cleavage pattern of that DNA. DNAs are compared
by looking for differences in restriction fragment lengths. A major
problem with this method (known as restriction fragment length
polymorphism mapping or RFLP mapping) is its inability to detect mutations
that do not affect cleavage with a restriction endonuclease. Thus, many
mutations are missed with this method. One study, by Jeffreys, Cell,
18:1-18 (1979), was able to detect only 0.7% of the mutational variants
estimated to be present in a 40,000 base pair region of human DNA. Another
problem is that the methods used to detect restriction fragment length
polymorphisms are very labor intensive, in particular, the techniques
involved with Southern blot analysis.
A technique for detecting specific mutations in any segment of DNA is
described in Wallace, et al., Nucl. Acids Res., 9:879-894 (1981). It
involves hybridizing the DNA to be analyzed (target DNA) with a
complementary, labeled oligonucleotide probe. Due to the thermal
instability of DNA duplexes containing even a single base pair mismatch,
differential melting temperature can be used to distinguish target DNAs
that are perfectly complementary to the probe from target DNAs that differ
by as little as a single nucleotide. In a related technique, described in
Landegren, et al., Science, 41:1077-1080 (1988), oligonucleotide probes
are constructed in pairs such that their junction corresponds to the site
on the DNA being analyzed for mutation. These oligonucleotides are then
hybridized to the DNA being analyzed. Base pair mismatch between either
oligonucleotide and the target DNA at the junction location prevents the
efficient joining of the two oligonucleotide probes by DNA ligase.
A. Nucleic acid hybridization
The base pairing of nucleic acids in a hybridization reaction forms the
basis of most nucleic acid analytical and diagnostic techniques. In
practice, tests based only on parameters of nucleic acid hybridization
function poorly in cases where the sequence complexity of the test sample
is high. This is partly due to the small thermodynamic differences in
hybrid stability, generated by single nucleotide changes, and the fact
that increasing specificity by lengthening the probe has the effect of
further diminishing this differential stability. Nucleic acid
hybridization is, therefore, generally combined with some other selection
or enrichment procedure for analytical and diagnostic purposes.
Combining hybridization with size fractionation of hybridized molecules as
a selection technique has been one general diagnostic approach. Size
selection can be carried out prior to hybridization. The best known prior
size selection technique is Southern Blotting (see Southern, E., Methods
in Enzymology, 69:152 (1980). In this technique, a DNA sample is subjected
to digestion with restriction enzymes which introduce double stranded
breaks in the phosphodiester backbone at or near the site of a short
sequence of nucleotides which is characteristic for each enzyme. The
resulting heterogeneous mixture of DNA fragments is then separated by gel
electrophoresis, denatured, and transferred to a solid phase where it is
subjected to hybridization analysis in situ using a labeled nucleic acid
probe. Fragments which contain sequences complementary to the labeled
probe are revealed visually or densitometrically as bands of hybridized
label. A variation of this method is Northern Blotting for RNA molecules.
Size selection has also been used after hybridization in a number of
techniques, in particular by hybrid protection techniques, by subjecting
probe/nucleic acid hybrids to enzymatic digestion before size analysis.
B. Polymerase extension of duplex primer:template complexes
Hybrids between primers and DNA targets can be analyzed by polymerase
extension of the hybrids. A modification of this methodology is the
polymerase chain reaction in which the purification is produced by
sequential hybridization reactions of anti-parallel primers, followed by
enzymatic amplification with DNA polymerase (see Saiki, et al., Science
239:487-491 (1988)). By selecting for two hybridization reactions, this
methodology provides the specificity lacking in techniques that depend
only upon a single hybridization reaction.
It has long been known that primer-dependent DNA polymerases have, in
general, a low error rate for the addition of nucleotides complementary to
a template. This feature is essential in biology for the prevention of
genetic mistakes which would have detrimental effects on progeny. The
specificity inherent in this enzymological reaction has been widely
exploited as the basis of the "Sanger" or dideoxy chain termination
sequencing methodology which is the ultimate nucleic acid typing
experiment. One type of Sanger DNA sequencing method makes use of mixtures
of the four deoxynucleoside triphosphates, which are normal DNA
precursors, and one of the four possible dideoxynucleoside triphosphates,
which have a hydrogen atom instead of a hydroxyl group attached to the 3'
carbon atom of the ribose sugar component of the nucleotide. DNA chain
elongation in the 5' to 3' direction ("downstream") requires this hydroxyl
group. As such, when a dideoxynucleotide is incorporated into the growing
DNA chain, no further elongation can occur. With one dideoxynucleotide in
the mixture, DNA polymerases can, from a primer:template combination,
produce a population of molecules of varying length, all of which
terminate after the addition of one out of the four possible nucleotides.
The series of four independent reactions, each with a different
dideoxynucleotide, generates a nested set of fragments, all starting at
the same 5' terminus of the priming DNA molecule and terminating at all
possible 3' nucleotide positions.
Another utilization of dideoxynucleoside triphosphates and a polymerase in
the analysis of DNA involves labeling the 3' end of a molecule. One
prominent manifestation of this technique provides the means for
sequencing a DNA molecule from its 3' end using the Maxam-Gilbert method.
In this technique, a molecule with a protruding 3' end is treated with
terminal transferase in the presence of radioactive dideoxy-ATP. One
radioactive nucleotide is added, rendering the molecule suitable for
sequencing. Both methods of DNA sequencing using labeled
dideoxynucleotides require electrophoretic separation of reaction products
in order to derive the typing information. Most methods require four
separate gel tracks for each typing determination.
The following two patents describe other methods of typing nucleic acids
which employ primer extension and labeled nucleotides. Mundy (U.S. Pat.
No. 4,656,127) describes a method whereby a primer is constructed
complementary to a region of a target nucleic acid of interest such that
its 3' end is close to a nucleotide in which variation can occur. This
hybrid is subject to primer extension in the presence of a DNA polymerase
and four deoxynucleoside triphosphates, one of which is an
.alpha.-thionucleotide. The hybrid is then digested using an exonuclease
enzyme which cannot use thio-derivatized DNA as a substrate for its
nucleolytic action (for example Exonuclease III of E. coli). If the
variant nucleotide in the template is complementary to one of the
thionucleotides in the reaction mixture, the resulting extended primer
molecule will be of a characteristic size and resistant to the
exonuclease; hybrids without thio-derivatized DNA will be digested. After
an appropriate enzyme digest to remove underivatized molecules, the
thio-derivatized molecule can be detected by gel electrophoresis or other
separation technology.
Vary and Diamond (U.S. Pat. No. 4,851,331) describes a method similar to
that of Mundy wherein the last nucleotide of the primer corresponds to the
variant nucleotide of interest. Since mismatching of the primer and the
template at the 3' terminal nucleotide of the primer is counterproductive
to elongation, significant differences in the amount of incorporation of a
tracer nucleotide will result under normal primer extension conditions.
This method depends on the use of a DNA polymerase, e.g., AMV reverse
transcriptase, that does not have an associated 3' to 5' exonuclease
activity.
The methods of Mundy and of Vary and Diamond have drawbacks. The method of
Mundy is useful but cumbersome due to the requirements of the second,
different enzymological system where the non-derivatized hybrids are
digested. The method of Vary is complicated by the fact that it does not
generate discrete reaction products. Any "false" priming will generate
significant noise in such a system which would be difficult to distinguish
from a genuine signal.
The present invention circumvents the problems associated with the methods
of Mundy and of Vary and Diamond for typing nucleic acid with respect to
particular nucleotides. With methods employing primer extension and a DNA
polymerase, the current invention will generate a discrete molecular
species one base longer than the primer itself. In many methods,
particularly those employing the polymerase chain reaction, the type of
reaction used to purify the nucleic acid of interest in the first step can
also be used in the subsequent detection step. Finally, with terminators
which are labeled with different detector moieties (for example different
fluorophors having different spectral properties), it will be possible to
use only one reagent for all sequence detection experiments. Furthermore,
if techniques are used to separate the terminated primers post-reaction,
sequence detection experiments at more than one locus can be carried out
in the same tube.
A recent article by Mullis (Scientific American, April 1990, pp. 56-65)
suggests an experiment, which apparently was not performed, to determine
the identity of a targeted base pair in a piece of double-stranded DNA.
Mullis suggests using four types of dideoxynucleosides triphosphate, with
one type of dideoxynucleoside triphosphate being radioactively labeled.
The present invention permits analyses of nucleic acid sequences that can
be useful in the diagnosis of infectious diseases, the diagnosis of
genetic disorders, and in the identification of individuals and their
parentage.
A number of methods have been developed for these purposes. Although
powerful, such methodologies have been cumbersome and expensive, generally
involving a combination of techniques such as gel electrophoresis,
blotting, hybridization, and autoradiography or non-isotopic revelation.
Simpler technologies are needed to allow the more widespread use of
nucleic acid analysis. In addition, tests based on nucleic acids are
currently among the most expensive of laboratory procedures and for this
reason cannot be used on a routine basis. Finally, current techniques are
not adapted to automated procedures which would be necessary to allow the
analysis of large numbers of samples and would further reduce the cost.
The current invention provides a method that can be used to diagnose or
characterize nucleic acids in biological samples without recourse to gel
electrophoretic size separation of the nucleic acid species. This feature
renders this process easily adaptable to automation and thus will permit
the analysis of large numbers of samples at relatively low cost. Because
nucleic acids are the essential blueprint of life, each organism or
individual can be uniquely characterized by identifiable sequences of
nucleic acids. It is, therefore, possible to identify the presence of
particular organisms or demonstrate the biological origin of certain
samples by detecting these specific nucleic acid sequences.
SUMMARY OF THE INVENTION
The subject invention provides a reagent composition comprising an aqueous
carrier and an admixture of at least two different terminators of a
nucleic acid template-dependent, primer extension reaction. Each of the
terminators is capable of specifically terminating the extension reaction
in a manner strictly dependent on the identity of the unpaired nucleotide
base in the template immediately adjacent to, and downstream of, the 3'
end of the primer. In addition, at least one of the terminators is labeled
with a detectable marker.
The subject invention further provides a reagent composition comprising an
aqueous carrier and an admixture of four different terminators of a
nucleic acid template-dependent, primer extension reaction. Each of the
terminators is capable of specifically terminating the extension reaction
as above and one, two, three, or four of the terminators is labeled with a
detectable marker.
The subject invention further provides a reagent as described above wherein
the terminators comprise nucleotides, nucleotide analogs,
dideoxynucleotides, or arabinoside triphosphates. The subject invention
also provides a reagent wherein the terminators comprise one or more of
dideoxyadenosine triphosphate (ddATP), dideoxycytosine triphosphate
(ddCTP), dideoxyguanosine triphosphate (ddGTP), dideoxythymidine
triphosphate (ddTTP), or dideoxyuridine triphosphate (ddUTP).
The subject invention also provides a method for determining the identity
of a nucleotide base at a specific position in a nucleic acid of interest.
First, a sample containing the nucleic acid of interest is treated, if
such nucleic acid is double-stranded, so as to obtain unpaired nucleotide
bases spanning the specific position. If the nucleic acid of interest is
single-stranded, this step is not necessary. Second, the sample containing
the nucleic acid of interest is contacted with an oligonucleotide primer
under hybridizing conditions. The oligonucleotide primer is capable of
hybridizing with a stretch of nucleotide bases present in the nucleic acid
of interest, immediately adjacent to the nucleotide base to be identified,
so as to form a duplex between the primer and the nucleic acid of interest
such that the nucleotide base to be identified is the first unpaired base
in the template immediately downstream of the 3' end of the primer in the
duplex of primer and the nucleic acid of interest. Enzymatic extension of
the oligonucleotide primer in the resultant duplex by one nucleotide,
catalyzed, for example, by a DNA polymerase, thus depends on correct base
pairing of the added nucleotide to the nucleotide base to be identified.
The duplex of primer and the nucleic acid of interest is then contacted
with a reagent containing four labeled terminators, each terminator being
labeled with a different detectable marker. The duplex of primer and the
nucleic acid of interest is contacted with the reagent under conditions
permitting base pairing of a complementary terminator present in the
reagent with the nucleotide base to be identified and the occurrence of a
template-dependent, primer extension reaction so as to incorporate the
terminator at the 3' end of the primer. The net result is that the
oligonucleotide primer has been extended by one terminator. Next, the
identity of the detectable marker present at the 3' end of the extended
primer is determined. The identity of the detectable marker indicates
which terminator has base paired to the next base in the nucleic acid of
interest. Since the terminator is complementary to the next base in the
nucleic acid of interest, the identity of the next base in the nucleic
acid of interest is thereby determined.
The subject invention also provides another method for determining the
identity of a nucleotide base at a specific position in a nucleic acid of
interest. This additional method uses a reagent containing four
terminators, only one of the terminators having a detectable marker.
The subject invention also provides a method of typing a sample of nucleic
acids which comprises identifying the base or bases present at each of one
or more specific positions, each such nucleotide base being identified
using one of the methods for determining the identity of a nucleotide base
at a specific position in a nucleic acid of interest as outlined above.
Each specific position in the nucleic acid of interest is determined using
a different primer. The identity of each nucleotide base or bases at each
position can be determined individually or the identities of the
nucleotide bases at different positions can be determined simultaneously.
The subject invention further provides a method for identifying different
alleles in a sample containing nucleic acids which comprises identifying
the base or bases present at each of one or more specific positions. The
identity of each nucleotide base is determined by the method for
determining the identity of a nucleotide base at a specific position in a
nucleic acid of interest as outlined above.
The subject invention also provides a method for determining the genotype
of an organism at one or more particular genetic loci which comprises
obtaining from the organism a sample containing genomic DNA and
identifying the nucleotide base or bases present at each of one or more
specific positions in nucleic acids of interest. The identity of each such
base is determined by using one of the methods for determining the
identity of a nucleotide base at a specific position in a nucleic acid of
interest as outlined above. The identities of the nucleotide bases
determine the different alleles and, thereby, determine the genotype of
the organism at one or more particular genetic loci.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-1C. Autoradiography of labeled DNA products after fractionation on
a polyacrylamide/urea gel. Panel A shows products of the "A" extension
reaction on oligonucleotide primer 182 directed by template
oligonucleotides 180 or 181. Panel B shows products of the "B" termination
reaction on oligonucleotide primer 182 annealed to template
oligonucleotides 180 or 181. Panel C shows the same products as in panel B
after purification on magnetic beads. Note: oligodeoxynucleotide 182 was
used as supplied by Midland Certified Reagents with no further
purification. The minor bands above and below the main band are presumably
contaminants due to incomplete reactions or side reactions that occurred
during the step-wise synthesis of the oligonucleotide. For a definition of
the "A" extension reaction and the "B" termination reaction, see "A.
GENERAL METHODS" in the Detailed Description of the Invention.
FIG. 2. Detection of Sequence Polymorphisms in PCR Products. Target
polymorphic DNA sequence showing amplification primers [SEQ ID NO:15] and
[SEQ ID NO:16], detection primers [SEQ ID NO:17] and [SEQ ID NO:18], and
molecular clone (plasmid) designations. For each primer, sites of binding
to one or the other strand of the target DNA sequence [SEQ ID NO:19] are
indicated by underlining, and the direction of DNA synthesis is indicated
by an arrow. Numbering for the target sequence is shown in the righthand
margin. Polymorphic sites at positions 114 and 190 are indicated by bold
lettering and a slash between the two polymorphic possibilities.
FIG. 3. Autoradiogram of gel-analyzed polymorphism test on PCR products.
Templates from PCR products of p183, p624, or p814 were analyzed with the
detection primers, TGL182 and TGL166, in a template-directed chain
extension experiment, as described in the specification. Reaction products
were fractionated by size on a polyacrylamide/urea DNA sequencing gel, and
incorporation of [.sup.35 S]-.alpha.-thio-dideoxy adenosine monophosphate
was assayed by autoradiography.
FIG. 4. Gel electrophoretic analysis of the labelled extension products of
primers TGL346 and TGL391. Productive primer-template complexes of TGL346
or TGL391 with the bead-bound oligonucleotide template, TGL382, were
subjected to primer extension labelling reactions with the four different | | |
