Digital amplification
DCFirst Claim
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1. A method for determining an allelic imbalance in a biological sample, comprising the steps of:
- 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 a selected genetic sequence on a first chromosome and a second number of assay samples which contain a reference genetic sequence on a second chromosome, wherein between 0.1 and 0.9 of the assay samples yield an amplification product;
comparing the first number of assay samples to the second number of assay samples to ascertain an allelic imbalance in the biological sample.
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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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22 Claims
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1. A method for determining an allelic imbalance in 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 a selected genetic sequence on a first chromosome and a second number of assay samples which contain a reference genetic sequence on a second chromosome, wherein between 0.1 and 0.9 of the assay samples yield an amplification product; comparing the first number of assay samples to the second number of assay samples to ascertain an allelic imbalance in the biological sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A method for determining an allelic imbalance in a biological sample, comprising the steps of:
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distributing nucleic acid template molecules from 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 a selected genetic sequence on a first chromosome and a second number of assay samples which contain a reference genetic sequence on a second chromosome; comparing the first number of assay samples to the second number of assay samples to ascertain an allelic imbalance between the first chromosome and the second chromosome in the biological sample. - View Dependent Claims (20, 21, 22)
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Specification