Copy number variation determination, methods and systems
AssigneeFluidigm Corporation
InventorRamesh Ramakrishnan
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Abstract
The present invention methods and systems for determining copy number variation of a target polynucleotide in a genome of a subject including amplification based techniques. Methods can include pre-amplification of the sample followed by distribution of sample and a plurality of reaction volumes, quantitative detection of a target polynucleotide and a reference polynucleotide, and analysis so as to determine the relative copy number of the target polynucleotide sequence in the genome of the subject.
Claims17
A method for determining the relative copy number of a target polynucleotide sequence in a genome of a subject, comprising:
- pre-amplifying a target gene sequence and a reference gene sequence in a sample containing genomic DNA of the subject;
- assaying the target gene sequence and the reference gene sequence of the pre-amplified sample by digital PCR;
- determining (a) the number of amplified polynucleotide molecules containing the target gene sequence and (b) the number of amplified polynucleotide molecules containing the reference gene sequence and determining the ratio of (a) to (b).
A method for determining a copy number of a target polynucleotide sequence in a genome of a subject, comprising:
- conducting a first polynucleotide amplification of a DNA sample obtained from a subject, wherein both a target polynucleotide sequence and a reference polynucleotide sequence, said reference sequence having a predetermined genomic copy number N, are amplified, thereby producing an amplified sample;
- distributing all or a portion of the amplified sample into a plurality of isolated reaction volumes;
- in each reaction volume conducting a second polynucleotide amplification in which the target polynucleotide sequence or a subsequence thereof is amplified if present and the reference polynucleotide sequence or a subsequent thereof is amplified if present;
- determining the number of reaction volumes in which the target polynucleotide sequence or subsequence thereof is present A and determining (b) the number of reaction volumes in which the reference polynucleotide sequence or subsequence thereof is present B;
- wherein the copy number of the target polynucleotide in the genome is approximately equal to (A)/(B)×N.
Dependencies are inferred from the claim text.