Digital amplification
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1. A method for detecting an imbalance in a mixed population of human genomic nucleic acid sequences, comprising:
- distributing a mixed population of cell-free, human genomic nucleic acid template molecules from a sample into a set comprising at least fifteen assay samples such that said at least fifteen assay samples each comprises less than ten template molecules, wherein the mixed population comprises at least a first and a second human genomic sequence, wherein the first sequence is on a first chromosome and the second sequence is on a second chromosome;
amplifying the template molecules in the assay samples, wherein an assay sample with a single template molecule forms homogeneous amplification products in the assay sample;
analyzing by determining nucleic acid sequence of amplification products in the assay samples of the set to determine a first number of assay samples in the set which contain the first human genomic sequence and a second number of assay samples in the set which contain the second human genomic sequence;
comparing the first number to the second number to determine an imbalance in the mixed population between the first and the second chromosomes.
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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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11 Claims
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1. A method for detecting an imbalance in a mixed population of human genomic nucleic acid sequences, comprising:
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distributing a mixed population of cell-free, human genomic nucleic acid template molecules from a sample into a set comprising at least fifteen assay samples such that said at least fifteen assay samples each comprises less than ten template molecules, wherein the mixed population comprises at least a first and a second human genomic sequence, wherein the first sequence is on a first chromosome and the second sequence is on a second chromosome; amplifying the template molecules in the assay samples, wherein an assay sample with a single template molecule forms homogeneous amplification products in the assay sample; analyzing by determining nucleic acid sequence of amplification products in the assay samples of the set to determine a first number of assay samples in the set which contain the first human genomic sequence and a second number of assay samples in the set which contain the second human genomic sequence; comparing the first number to the second number to determine an imbalance in the mixed population between the first and the second chromosomes. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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