Methods for determining pre-amplification levels of a nucleic acid target sequence from post-amplification levels of product
First Claim
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1. A method for determining the amount of a target nucleic acid sequence present in a sample, comprising:
- a) contactingI) a sample containing one or more copies of said target sequence, andii) known amounts of at least one standard nucleic acid with oligonucleotide primers, reagents, a reverse transcriptase activity and an RNA polymerase activity under conditions sufficient to cause a transcription-mediated amplification of said target sequence, if present, under said conditions,b) amplifying said standard nucleic acid and said target sequence to produce a standard-specific RNA amplicon and a target-specific RNA amplicon,c) measuring the amount of each said standard-specific amplicons and specific amplicons and said target-specific amplicons produced, andd) correlating the amount of standard-specific amplicon reaction products with the known pre-amplification amounts of said standard nucleic acid to produce a standard curve, ande) determining the pre-amplification amount of the target sequence by comparing the measurement of target-specific amplicons made at step c) with said standard curve,wherein no RNAse H additional to that provided by said reverse transcriptase is used.
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Abstract
Methods and kits for relating initial amounts of target nucleic acids present in a sample to target-specific amplification products. It has been discovered that the transcription-mediated amplification system is capable of producing a quantitative relationship between target input and target-specific output. Further, the present invention relates to methods for carefully controlling this relationship resulting in an unexpectedly high degree of reproducability. Also described are useful methods for extending the dynamic range of transcription-based amplification systems.
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Citations
40 Claims
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1. A method for determining the amount of a target nucleic acid sequence present in a sample, comprising:
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a) contacting I) a sample containing one or more copies of said target sequence, and ii) known amounts of at least one standard nucleic acid with oligonucleotide primers, reagents, a reverse transcriptase activity and an RNA polymerase activity under conditions sufficient to cause a transcription-mediated amplification of said target sequence, if present, under said conditions, b) amplifying said standard nucleic acid and said target sequence to produce a standard-specific RNA amplicon and a target-specific RNA amplicon, c) measuring the amount of each said standard-specific amplicons and specific amplicons and said target-specific amplicons produced, and d) correlating the amount of standard-specific amplicon reaction products with the known pre-amplification amounts of said standard nucleic acid to produce a standard curve, and e) determining the pre-amplification amount of the target sequence by comparing the measurement of target-specific amplicons made at step c) with said standard curve, wherein no RNAse H additional to that provided by said reverse transcriptase is used. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method for determining the amount of a target nucleic acid sequence present in a sample, comprising:
- incubating a reaction mixture containing a DNA-directed DNA polymerase activity, an RNA-directed DNA polymerase activity, an RNAse H activity, a RNA polymerase activity, at least one promoter-primer and said target sequence under conditions sufficient to cause a transcription-mediated nucleic acid amplification reaction, measuring the amount of a target-specific amplicon produced in said reaction, and relating the amount of said amplicon to the amount of target originally present in the sample, wherein no RNAse H additional to that provided by said reverse transcriptase is used.
- View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34)
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31. A method for determining the amount of a target nucleic acid sequence present in a sample, comprising:
- incubating a reaction mixture containing a DNA-directed DNA polymerase activity, an RNA-directed DNA polymerase activity, an RNAse H activity, a RNA polymerase activity, at least one promoter-primer and said target sequence under conditions sufficient to cause a transcription-mediated nucleic acid amplification reaction, measuring the amount of a target-specific amplicon produced in said reaction, and relating the amount of said amplicon to the amount of target originally present in the sample, wherein;
said relating step comprises comparing the amount of said amplicon to a standard curve correlating a known preamplification amount of a standard nucleic acid with a post-amplification amount of a corresponding standard-specific amplicon;
said conditions are made suboptimal for achieving a standard amplification reaction rate; and
said suboptimal condition is caused by the addition of an amount of a chelating agent to said reaction mixture, wherein said amount does not cause complete inhibition of said amplification reaction.
- incubating a reaction mixture containing a DNA-directed DNA polymerase activity, an RNA-directed DNA polymerase activity, an RNAse H activity, a RNA polymerase activity, at least one promoter-primer and said target sequence under conditions sufficient to cause a transcription-mediated nucleic acid amplification reaction, measuring the amount of a target-specific amplicon produced in said reaction, and relating the amount of said amplicon to the amount of target originally present in the sample, wherein;
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35. A method for determining the amount of a target nucleic acid sequence present in a sample, comprising:
- incubating a reaction mixture containing a DNA-directed DNA polymerase activity, an RNA-directed DNA polymerase activity, an RNAse H activity, a RNA polymerase activity, at least one promoter-primer and said target sequence under conditions sufficient to cause a transcription-mediated nucleic acid amplification reaction, measuring the amount of a target-specific amplicon produced in said reaction, and relating the amount of said amplicon to the amount of target originally present in the sample, wherein;
said relating step comprises comparing the amount of said amplicon to a standard curve correlating a known pre-amplification amount of a standard nucleic acid with a post-amplification amount of a corresponding standard-specific amplicon;
said conditions are made suboptimal for achieving a standard amplification reaction rate, and at least one promoter-primer is modified to have a reduced ability to initiate transcription of said target sequence relative to an unmodified promoter-primer. - View Dependent Claims (36, 37, 38, 39)
- incubating a reaction mixture containing a DNA-directed DNA polymerase activity, an RNA-directed DNA polymerase activity, an RNAse H activity, a RNA polymerase activity, at least one promoter-primer and said target sequence under conditions sufficient to cause a transcription-mediated nucleic acid amplification reaction, measuring the amount of a target-specific amplicon produced in said reaction, and relating the amount of said amplicon to the amount of target originally present in the sample, wherein;
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40. A method for determining the amount of a target nucleic acid sequence present in a sample comprising the steps:
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a) making serial dilutions of said sample, b) contacting said serially diluted sample with oligonucleotide primers, reagents, a reverse transcriptase activity and an RNA polymerase activity under conditions sufficient to promote a transcription-mediated amplification reaction, c) detecting a target-specific RNA amplicon, if present, in reaction mixtures corresponding to said dilutions, and d) correlating a dilution factor corresponding to the least diluted sample at which no target-specific amplicon was detected with a number of target molecules originally in the undiluted sample as an estimation of said amount, wherein no RNAse H additional to that provided by said reverse transcriptase is used.
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Specification