DIGITAL AMPLIFICATION
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.
1 . (canceled)
2 . A method for detecting an imbalance in a sample of genomic nucleic acid sequences, comprising:
distributing or diluting cell-free, genomic nucleic acid template molecules from the sample into a set of at least fifteen assay samples such that each assay sample comprises less than ten template molecules, wherein the sample comprises at least a first genomic sequence on a first chromosome and a second genomic sequence on a second chromosome;
amplifying the first and second genomic sequences in the assay samples, such that assay samples with a single template molecule individually form homogeneous amplification products;
determining the nucleic acid sequence of the amplification products to determine a ratio that corresponds to the relative amounts of the first and second genomic sequences in the sample, wherein said ratio reflects an imbalance between the first and the second chromosomes in the sample.
3 . The method of claim 2 , wherein the assay samples of the set have on average 0.5 molecules of template.
4 . The method of claim 2 , wherein between 0.1 and 0.9 of the assay samples yield an amplification product.
5 . The method of claim 2 , wherein the sample of genomic nucleic acid sequences is distributed or diluted to a single template molecule level in the assay samples.
6 . The method of claim 2 , wherein the sample of genomic nucleic acid sequences is from a blood or tissue sample.
7 . The method of claim 2 , wherein the sample of genomic nucleic acid sequences is distributed or diluted such that at least twenty-five assay samples each comprise less than ten template molecules.
8 . The method of claim 2 , wherein the sample of genomic nucleic acid sequences is distributed or diluted such that at least one hundred assay samples each comprise less than ten template molecules.
9 . The method of claim 2 , wherein the sample of genomic nucleic acid sequences is distributed or diluted such that at least five hundred assay samples each comprise less than ten template molecules.
10 . The method of claim 2 , wherein the sample of genomic nucleic acid sequences is distributed or diluted such that at least one thousand assay samples each comprise less than ten template molecules.
11 . The method of claim 2 , wherein the sample of genomic nucleic acid sequences is distributed or diluted such that at least five hundred assay samples are distributed or diluted to a single template molecule level.
12 . The method of claim 2 , wherein the sample of genomic nucleic acid sequences is distributed or diluted such that at least one thousand assay samples are distributed or diluted to a single template molecule level.
13 . The method of claim 2 , wherein the sample of genomic nucleic acid sequences is distributed or diluted such that at least five hundred assay samples each have on average 0.5 molecules of template.
14 . The method of claim 2 , wherein the sample of genomic nucleic acid sequences is distributed or diluted such that at least one thousand assay samples each have on average 0.5 molecules of template.
15 . The method of claim 2 , wherein the sample of genomic nucleic acid sequences is distributed or diluted such that between 0.1 and 0.9 of at least one thousand assay samples yield an amplification product.
16 . The method of claim 2 , wherein the sample of genomic nucleic acid sequences is distributed or diluted such that at least five hundred assay samples each have one template molecule.
17 . The method of claim 2 , wherein the first genomic sequence, the second genomic sequence, or both are non-polymorphic markers.
18 . A method for detecting an imbalance in a sample of genomic nucleic acid sequences, comprising:
distributing or diluting cell-free, genomic nucleic acid template molecules from the sample into a set of at least fifteen assay samples such that said at least fifteen assay samples each comprise an average of 0.5 molecules of template per assay sample, wherein the sample comprises at least a first genomic sequence on a first chromosome and a second genomic sequence on a second chromosome;
amplifying the first and second genomic sequences in the assay samples, such that assay samples with a single template molecule individually form homogeneous amplification products;
determining the nucleic acid sequence of amplification products for the assay samples to determine a ratio that corresponds to the relative amounts of the first and second genomic sequences in the sample, wherein said ratio reflects an imbalance between the first and the second chromosomes in the sample.
19 . The method of claim 18 , wherein between 0.1 and 0.9 of the assay samples yield an amplification product.
20 . The method of claim 18 , wherein the sample of genomic nucleic acid sequences is from a blood or tissue sample.
21 . The method of claim 18 , wherein the first genomic sequence, the second genomic sequence, or both are non-polymorphic markers.