Method for determining nucleotide identity through primer extension
DCFirst Claim
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1. 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 the 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 which is fully complementary to and which 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 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; and
(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 immediately downstream of the 3'"'"' end of the 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 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.
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Abstract
The invention concerns a reagent composition that employs at least two different terminators of a nucleic acid template-dependent primer extension reaction to determine the identity of a nucleotide base at a specific position in a nucleic acid of interest. The invention also concerns the method for determining such identification. The invention may be used to determine the presence or absence of a specific nucleotide sequence in a sample. It may also be employed in determination of genotype and in the identification of different alleles.
365 Citations
48 Claims
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1. A method of determining the identity of a nucleotide base at a specific position in a nucleic acid of interest, which comprises:
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(a) treating a sample containing the nucleic acid of interest, if the 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 which is fully complementary to and which 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 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; and (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 immediately downstream of the 3'"'"' end of the 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 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 48)
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43. A method of determining the presence or absence of a particular target nucleic acid molecule in a sample of nucleic acids, which comprises:
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(a) treating the 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 which is fully complementary to and which hybridizes specifically to a stretch of nucleotide bases present in said target nucleic acid molecule immediately adjacent to a preselected nucleotide base, under high stringency hybridization conditions so as to form a duplex between the primer and the nucleic acid of interest such that the preselected nucleotide base is the first unpaired base in the template immediately downstream of the 3'"'"' end of the primer in said duplex; and (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 immediately downstream of the 3'"'"' end of the 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 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 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 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, and wherein said detection is conducted in the absence of non-terminator nucleotides. - View Dependent Claims (44, 45)
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46. A method of determining the identity of a nucleotide base at a specific position in a nucleic acid of interest which comprises:
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(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 which is fully complementary to and which 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 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; and (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 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 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 market 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.
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47. A method of determining the presence or absence of a particular target nucleic acid molecule in a sample of nucleic acids which comprises:
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(a) (1) incubating said 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 desired polynucleotide if said desired polynucleotide is present in said sample, wherein said incubation is conducted under conditions sufficient to permit said amplification to occur; (2) treating said incubated 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 double-stranded nucleic acids; (b) contacting the sample from step (a2), under hybridizing conditions, with an oligonucleotide primer which is fully complementary to and which hybridizes specifically to a stretch of nucleotide bases present in said target nucleic acid molecule immediately adjacent to a preselected nucleotide base, so as to form a duplex between the primer and the nucleic acid of interest such that the preselected nucleotide base is the first unpaired base in the template immediately downstream of the 3'"'"' end of the primer in said duplex; and (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 immediately downstream of the 3'"'"' end of the 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 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 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; 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, and wherein said detection is conducted in the absence of non-terminator nucleotides.
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Specification