Methods for relatvie 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 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
b) 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, wherein said primer extension reaction is performed using a sequential dispensation order of individual nucleotides, such that primer extension reactions of said respective target and control amplicons are performed sequentially of each other, and wherein determining relative quantities of a primer extension reaction comprises 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.
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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.
8 Citations
39 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 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
b) 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, wherein said primer extension reaction is performed using a sequential dispensation order of individual nucleotides, such that primer extension reactions of said respective target and control amplicons are performed sequentially of each other, and wherein determining relative quantities of a primer extension reaction comprises 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, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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- 7. The method of claim 65 wherein said pyrophosphate is detected enzymatically using the enzyme luciferase as a PPi-detection enzyme.
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17. The method of 16, wherein said gene or fragment thereof is a gene selected from the group consisting of a disease resistance gene, an antibiotic resistance gene and a gene conferring an investigated trait.
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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 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
b) 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, wherein said primer extension reaction is performed using a sequential dispensation order of individual nucleotides, such that primer extension reactions of said respective target and control amplicons are performed sequentially of each other, and wherein determining relative quantities of a primer extension reaction comprises 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)
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- 21. The method of 20, wherein said gene or fragment thereof is a gene selected from the group consisting of a disease resistance gene, an antibiotic resistance gene, and a gene conferring an investigated trait.
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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 a target nucleic acid sequence from said plasmid 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
b) 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, wherein said primer extension reaction is performed using a sequential dispensation order of individual nucleotides, such that primer extension reactions of said respective target and control amplicons are performed sequentially of each other, and wherein determining relative quantities of a primer extension reaction comprises 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 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 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;
b) 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, wherein said primer extension reaction is performed using a sequential dispensation order of individual nucleotides, such that primer extension reactions of said respective target and control amplicons are performed sequentially of each other, and wherein determining relative quantities of a primer extension reaction comprises 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, and c) determining if said copy number of said target nucleic acid sequence is one, wherein a copy number of said target nucleic acid sequence equal to one is indicative of an organism having a single copy of said target nucleic acid sequence. - View Dependent Claims (25, 26)
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- 27. The method of 26, wherein said gene or fragment thereof is a gene selected from the group consisting of a disease resistance gene, an antibiotic resistance gene, and a gene conferring an investigated trait.
Specification