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. 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.
2. The method of claim 1 wherein the set comprises at least 500 assay samples.
3. The method of claim 1 wherein the set comprises at least 1000 assay samples.
4. The method of claim 1 wherein the amplified molecules in each of the assay samples within the first and second numbers of assay samples are homogeneous such that the first number of assay samples do not contain the second human genomic sequence and the second number of assay samples do not contain the first human genomic sequence.
5. The method of claim 1 wherein the distributing is performed by diluting.
6. The method of claim 1 wherein the biological sample is from blood.
7. The method of claim 1 wherein the first human genomic sequence is a non-polymorphic marker.
8. The method of claim 1 wherein the mixed population of nucleic acid template molecules is distributed such that at least one thousand assay samples are distributed to a single template molecule level.
9. The method of claim 1 wherein the mixed population of nucleic acid template molecules is distributed such that at least one thousand assay samples each comprise less than ten template molecules.
10. The method of claim 1 wherein the mixed population of nucleic acid template molecules is distributed such that at least five hundred assay samples each comprise less than ten template molecules.
11. The method of claim 1 wherein the mixed population of nucleic acid template molecules is distributed such that at least one hundred assay samples each comprise less than ten template molecules.