Copy number variation determination, methods and systems

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.

What is claimed is: 
     
         1 . 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).   
     
     
         2 . The method of  claim 1  wherein the sample is from a human. 
     
     
         3 . The method of  claim 1  wherein the ratio of (a) to (b) is about 0.5 and there is a deletion of (a) on one chromosome. 
     
     
         4 . The method of  claim 1  wherein the ratio of (a) to (b) is about 1.5 and there is a duplication of (a) on one chromosome. 
     
     
         5 . 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.   
     
     
         6 . The method of  claim 2 , wherein conducting the first polynucleotide amplification comprises combining the biological sample with a composition comprising primers specific for the target polynucleotide sequence and primers specific for reference polynucleotide sequence, and conducting a polymerase chain reaction (PCR) assay so as to separately amplify target polynucleotide and reference polynucleotide in substantially equal proportion. 
     
     
         7 . The method of  claim 6  wherein the first polynucleotide amplification comprises from 4 to 15 cycles. 
     
     
         8 . The method of  claim 2 , wherein the reaction volumes are disposed in a microfluidic device, and the first polynucleotide amplification is conducted in a reaction volume separate from the microfluidic device. 
     
     
         9 . The method of  claim 2 , wherein prior to the step of distributing, all or a portion of the amplified sample is combined with reagents selected for quantitative amplification of target gene sequence and reference gene sequence. 
     
     
         10 . The method of  claim 9  wherein the reference gene sequence amplification primers used in the first polynucleotide amplification step are the same as those used in the second polynucleotide amplification step. 
     
     
         11 . The method of  claim 10  wherein the target gene sequence amplification primers used in the first polynucleotide amplification step are the same as those used in the second polynucleotide amplification step. 
     
     
         12 . The method of  claim 9 , wherein the reagents comprise a first probe that selectively hybridizes to a target gene sequence and a second probe that selectively hybridizes to a reference gene sequence under conditions suitable for polynucleotide amplification. 
     
     
         13 . The method of  claim 12 , wherein the first and second probes comprise different detectable labels, and wherein binding of the first or second probe or degradation of the first or second probe upon polymerase chain reaction (PCR) based polymerization results in a change in detectable fluorescence of the respective detectable label. 
     
     
         14 . The method of  claim 1 , wherein the reference gene sequence comprises a polynucleotide sequence at least partially encoding an RNaseP enzyme, beta-actin or GAPDH. 
     
     
         15 . The method of  claim 1  or  claim 2 , wherein a ratio of target gene sequence to reference gene sequence substantially deviating from a value of 1 indicates an abnormal target gene sequence copy number in the genome of the patient. 
     
     
         16 . The method of  claim 1  or  claim 2 , wherein determining the relative copy number of the target gene sequence comprises detecting a loss of heterozygosity in the genome of the subject. 
     
     
         17 . The method of  claim 1  or  claim 2 , wherein a ratio of target gene sequence to reference gene sequence with a value substantially greater than or less than 1 indicates a loss of heterozygosity in the genome of the patient.