Method for determining nucleotide identity through extension of immobilized primer
DCFirst Claim
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.
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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 an immobilized 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.
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Citations
56 Claims
- 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.
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13. 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 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. - View Dependent Claims (17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56)
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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:
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(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. - View Dependent Claims (19)
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15. 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, 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.
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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:
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(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.
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Specification