Methods for relative quantification of specific nucleic acid sequences
First Claim
1. A method of assessing an amount of a known target nucleic acid sequence in a sample comprising:
- a) co-amplifying using a duplex PCR said known target nucleic acid sequence and a known amount of a known control nucleic acid sequence using different target and control specific primers to produce respective target and control amplicons, wherein said control nucleic acid sequence is different than said target nucleic acid sequence, and wherein said co-amplifying is stopped during an exponential phase of the duplex PCR;
b) predetermining a dispensation order of nucleotides based on the nucleotide sequence order of the target nucleic acid and control nucleic acid sequence;
c) dispensing the nucleotides in a single reaction in the predetermined dispensation order in a primer extension reaction such that the target and control nucleic acids are extended sequentially; and
d) determining relative amounts of said target and control amplicons bycomparing a quantity of nucleotides incorporated during said primer extension reaction for said target amplicon with a quantity of nucleotides incorporated during said primer extension reaction for said control amplicon, wherein relative amounts of said respective amplicons are proportional to relative quantities of nucleotides incorporated during said primer extension reactions and said amount of said target nucleic acid sequence in said sample is proportional thereto.
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Abstract
This invention relates generally to a method for quantifying the number of occurrences of a specific nucleic acid sequence within a nucleic acid sample in order to circumvent the shortcomings of the methods currently available and to provide reliable quantification of a specific nucleic acid sequence within a nucleic acid sample. The present invention provides a method of assessing an amount of a known target nucleic acid sequence in a sample comprising co-amplifying said target nucleic acid sequence and a known amount of a known control nucleic acid sequence to produce respective target and control amplicons, wherein said control nucleic acid sequence is different than said target nucleic acid sequence; and determining relative amounts of said respective amplicons by determining relative quantities of a primer extension reaction using each of said respective amplicons as a template.
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Citations
35 Claims
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1. A method of assessing an amount of a known target nucleic acid sequence in a sample comprising:
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a) co-amplifying using a duplex PCR said known target nucleic acid sequence and a known amount of a known control nucleic acid sequence using different target and control specific primers to produce respective target and control amplicons, wherein said control nucleic acid sequence is different than said target nucleic acid sequence, and wherein said co-amplifying is stopped during an exponential phase of the duplex PCR; b) predetermining a dispensation order of nucleotides based on the nucleotide sequence order of the target nucleic acid and control nucleic acid sequence; c) dispensing the nucleotides in a single reaction in the predetermined dispensation order in a primer extension reaction such that the target and control nucleic acids are extended sequentially; and d) determining relative amounts of said target and control amplicons by comparing a quantity of nucleotides incorporated during said primer extension reaction for said target amplicon with a quantity of nucleotides incorporated during said primer extension reaction for said control amplicon, wherein relative amounts of said respective amplicons are proportional to relative quantities of nucleotides incorporated during said primer extension reactions and said amount of said target nucleic acid sequence in said sample is proportional thereto. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 31, 32, 33, 34, 35)
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18. A method of assessing copy number of a target nucleic acid sequence in a genome of an organism comprising:
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a) co-amplifying using a duplex PCR said target nucleic acid sequence and a known amount of a known control nucleic acid sequence using different target and control specific primers to produce respective target and control amplicons, wherein said control nucleic acid sequence is different than said target nucleic acid sequence, wherein said co-amplifying is stopped during an exponential phase of the duplex PCR; b) predetermining a dispensation order of nucleotides based on the nucleotide sequence order of the target nucleic acid and control nucleic acid sequence; c) dispensing the nucleotides in a single reaction in the predetermined dispensation order in a primer extension reaction such that the target and control nucleic acids are extended sequentially; and d) determining relative amounts of said target and control amplicons by comparing a quantity of nucleotides incorporated during said primer extension reaction for said target amplicon with a quantity of nucleotides incorporated during said primer extension reaction for said control amplicon, wherein relative amounts of said respective amplicons are proportional to relative quantities of nucleotides incorporated during said primer extension reactions and copy number of said target nucleic acid sequence in said genome is proportional thereto. - View Dependent Claims (19, 20, 21, 23)
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22. A method of assessing copy number of a plasmid in a cell comprising:
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a) co-amplifying using a duplex PCR a target nucleic acid sequence from said plasmid and a known amount of a known control nucleic acid sequence using different target and control specific primers to produce respective target and control amplicons, wherein said control nucleic acid sequence is different than said target nucleic acid sequence, wherein said co-amplifying during an exponential phase of the duplex PCR; b) predetermining a dispensation order of nucleotides based on the nucleotide sequence order of the target nucleic acid and control nucleic acid sequence; c) dispensing the nucleotides in a single reaction in the predetermined dispensation order in a primer extension reaction such that the target and control nucleic acids are extended sequentially; and d) determining relative amounts of said target and control amplicons by comparing a quantity of nucleotides incorporated during said primer extension reaction for said target amplicon with a quantity of nucleotides incorporated during said primer extension reaction for said control amplicon, wherein the relative amounts of said respective amplicons are proportional to the relative quantities of nucleotides incorporated during said primer extension reactions and said copy number of said plasmid in said cell is proportional thereto.
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24. A method of identifying an organism having a single copy of a target nucleic acid sequence comprising:
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a) co-amplifying using a duplex PCR said target nucleic acid sequence and a known amount of a known control nucleic acid sequence using different target and control specific primers to produce respective target and control amplicons, wherein said control nucleic acid sequence is different than said target nucleic acid sequence, wherein said co-amplifying is stopped during an exponential phase of the duplex PCR; b) predetermining a dispensation order of nucleotides based on the nucleotide sequence order of the target nucleic acid and control nucleic acid sequence; c) dispensing the nucleotides in a single reaction in the predetermined dispensation order in a primer extension reaction such that the target and control nucleic acids are extended sequentially; d) determining relative amounts of said target and control amplicons by comparing a quantity of nucleotides incorporated during said primer extension reaction for said target amplicon with a quantity of nucleotides incorporated during said primer extension reaction for said control amplicon, wherein relative amounts of said respective amplicons are proportional to relative quantities of nucleotides incorporated during said primer extension reactions and said copy number of said target nucleic acid sequence is proportional thereto, wherein if a copy number of said target nucleic acid sequence is equal to one, it is indicative of an organism having a single copy of said target nucleic acid sequence. - View Dependent Claims (25, 26, 27, 28, 29, 30)
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Specification