Digital amplification
First Claim
1. A method for detecting a cancer-associated mutant nucleic acid that is present in a patient sample at a low level relative to a corresponding wild-type nucleic acid, the method comprising:
- diluting nucleic acids in a biological sample to form a set comprising a plurality of assay samples;
amplifying the nucleic acids in the assay samples to form a population of amplified molecules;
performing an assay on the amplified molecules in each assay sample to determine whether a cancer-associated mutation is present in at least one of the assay samples;
wherein the step of diluting in performed until at least one-fiftieth of the assay samples in the set comprise a number (N) of molecules such that 1/N is larger than a ratio of the mutant nucleic acid to the wild-type nucleic acid required to detect the mutant nucleic acid if it is present in the assay sample.
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Accused Products
Abstract
The identification of pre-defined mutations expected to be present in a minor fraction of a cell population is important for a variety of basic research and clinical applications. The exponential, analog nature of the polymerase chain reaction is transformed into a linear, digital signal suitable for this purpose. Single molecules can be isolated by dilution and individually amplified; each product is then separately analyzed for the presence of pre-defined mutations. The process provides a reliable and quantitative measure of the proportion of variant sequences within a DNA sample.
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Citations
48 Claims
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1. A method for detecting a cancer-associated mutant nucleic acid that is present in a patient sample at a low level relative to a corresponding wild-type nucleic acid, the method comprising:
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diluting nucleic acids in a biological sample to form a set comprising a plurality of assay samples; amplifying the nucleic acids in the assay samples to form a population of amplified molecules; performing an assay on the amplified molecules in each assay sample to determine whether a cancer-associated mutation is present in at least one of the assay samples; wherein the step of diluting in performed until at least one-fiftieth of the assay samples in the set comprise a number (N) of molecules such that 1/N is larger than a ratio of the mutant nucleic acid to the wild-type nucleic acid required to detect the mutant nucleic acid if it is present in the assay sample. - 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)
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29. A method for determining the ratio of a selected genetic sequence in a population of genetic sequences from a blood sample, comprising the steps of:
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diluting nucleic acid template molecules from a blood sample to form a set comprising a plurality of assay samples; amplifying the template molecules within the assay samples to form a population of amplified molecules in the assay samples of the set; analyzing the amplified molecules in the assay samples of the set to determine a first number of assay samples which contain the selected genetic sequence and a second number of assay samples which contain a reference genetic sequence; comparing the first number to the second number to ascertain a ratio which reflects the composition of the blood sample. - View Dependent Claims (30, 31, 32, 33, 34)
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35. A method for determining the ratio of a selected non-polymorphic marker in a population of genetic sequences in a biological sample, comprising the steps of:
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diluting nucleic acid template molecules in a biological sample to form a set comprising a plurality of assay samples; amplifying the template molecules within the assay samples to form a population of amplified molecules in the assay samples of the set; analyzing the amplified molecules in the assay samples of the set to determine a first number of assay samples which contain the selected non-polymorphic marker and a second number of assay samples which contain a reference non-polymorphic marker, wherein the selected and reference non-polymorphic markers are on distinct chromosomes; comparing the first number to the second number to ascertain a ratio which reflects the composition of the biological sample; and identifying an allelic imbalance based on the ratio ascertained. - View Dependent Claims (36, 37, 38)
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39. A method for determining the ratio of a selected genetic sequence in a population of genetic sequences from a blood sample, comprising the steps of:
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amplifying template molecules within a set comprising a plurality of assay samples to form a population of amplified molecules in each of the assay samples of the set, wherein the template molecules are obtained from a blood sample; analyzing the amplified molecules in the assay samples of the set to determine a first number of assay samples which contain the selected genetic sequence and a second number of assay samples which contain a reference genetic sequence, wherein at least one-fiftieth of the assay samples in the set comprise a number (N) of molecules such that 1/N is larger than the ratio of selected genetic sequences to total genetic sequences required to determine the presence of the selected genetic sequence; comparing the first number to the second number to ascertain a ratio which reflects the composition of the blood sample. - View Dependent Claims (40, 41, 42, 43, 44)
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45. A method for determining the ratio of a selected non-polymorphic marker in a population of non-polymorphic markers from a biological sample, comprising the steps of:
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amplifying template molecules within a set comprising a plurality of assay samples to form a population of amplified molecules in each of the assay samples of the set, wherein the template molecules are obtained from a biological sample; analyzing the amplified molecules in the assay samples of the set to determine a first number of assay samples which contain the selected non-polymorphic marker and a second number of assay samples which contain a reference non-polymorphic marker, wherein at least one-fiftieth of the assay samples in the set comprise a number (N) of molecules such that 1/N is larger than the ratio of selected non-polymorphic marker to total non-polymorphic markers required to determine the presence of the selected non-polymorphic marker, wherein the selected genetic sequence and the reference genetic sequence are on distinct chromosomes; comparing the first number to the second number to ascertain a ratio which reflects the composition of the biological sample; and identifying an allelic imbalance based on the ratio ascertained. - View Dependent Claims (46, 47, 48)
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Specification