Array-based methods for analysing mixed samples using differently labelled allele-specific probes

Abstract

This disclosure provides methods and kits useful in analysis of mixed nucleic acid populations, including for multiplex genotyping of a mixed nucleic acid sample and for detecting differences in copy number of a target polynucleotide and/or a target chromosome (e.g., microdeletions, duplications, and aneuploidies). The disclosure also provides methods and systems useful in the diagnosis of genetic abnormalities in a mixed nucleic acid population taken non-invasively from an organism, such as a sample of blood, plasma, serum, urine stool or saliva. The disclosed methods and systems find use in multiple applications, including prenatal testing and cancer diagnostics. The method is based on the hybridization of amplified fragments obtained from the sample, e.g., using molecular inversion probes (MIP) to an oligonucleotide array and the detection of the alleles based on different signals from the different alleles of the SNP.

1 . A kit for the detection of fetal copy number variation, comprising:
 a) a capture device having a plurality of nucleic acid fragments corresponding to at least one chromosomal target region attached thereto;   b) a plurality of molecular probes capable of hybridizing to a mixed nucleic acid population that includes a major subpopulation and a minor subpopulation, wherein the major and minor subpopulations each include a target sequence located in a first chromosomal region and containing a polymorphic site, wherein the polymorphic site includes combinations of a first nucleotide variant and a second nucleotide variant; and   c) instructions for genotyping and detecting the polymorphic site.   
     
     
         2 . The kit of  claim 1 , wherein the capture device is a microarray. 
     
     
         3 . The kit of  claim 1 , wherein the chromosomal target region is on one or more of chromosomes 1, 5, 13, 18, 21, X, and Y. 
     
     
         4 . The kit of  claim 1 , wherein molecular probes are designed to genotype a single nucleotide polymorphism on one or more of chromosomes 1, 5, 13, 18, 21, X, and Y. 
     
     
         5 . The kit of  claim 1 , wherein the plurality of molecular probes comprises linear molecular inversion probes. 
     
     
         6 . The kit of  claim 5 , further comprising a first set of nucleotides and a second set of nucleotides for mixing with respective first and second channels of a nucleic acid sample following addition of the linear molecular inversion probes to the nucleic acid sample, wherein the first and second sets of nucleotides include different nucleotides. 
     
     
         7 . The kit of  claim 6 , wherein the first set of nucleotides comprises a mixture of dATP and dTTP, and is substantially free of dGTP and dCTP, and wherein the second set of nucleotides comprises a mixture of dGTP and dCTP, and is substantially free of dATP and dTTP. 
     
     
         8 . The kit of  claim 5 , further comprising a ligase for forming circularized probe compositions from the linear molecular inversion probes. 
     
     
         9 . The kit of  claim 8 , further comprising an exonuclease for cleaving linear molecular inversion probes and nucleic acid fragments following formation of the circularized probe compositions. 
     
     
         10 . The kit of  claim 8 , further comprising a restriction enzyme or a uracil-N-glycosylase for cleaving the circularized probe compositions to form linearized probe compositions. 
     
     
         11 . The kit of  claim 10 , further comprising primers and a polymerase configured to enable amplification of linearized probe compositions prior to contacting the linearized probe compositions to the capture device. 
     
     
         12 . The kit of  claim 1 , further comprising a detector for detecting signals indicative of presence or absence of the first nucleotide variant and the second nucleotide variant. 
     
     
         13 . A system for analyzing a mixed nucleic acid sample obtained from an organism, comprising:
 a plurality of linear molecular inversion probes capable of hybridizing to a mixed nucleic acid population that includes a major subpopulation and a minor subpopulation, wherein the major and minor subpopulations each include a target sequence located in a first chromosomal region and containing a polymorphic site, wherein the polymorphic site includes combinations of a first nucleotide variant and a second nucleotide variant;   a ligase for forming circularized probe compositions from the linear molecular inversion probes;   a first set of nucleotides; and   a second set of nucleotides different from the first set of nucleotides;   
     
     
         14 . The system of  claim 13 , wherein the capture device is a microarray. 
     
     
         15 . The system of  claim 13 , wherein the first set of nucleotides comprises a mixture of dATP and dTTP, and is substantially free of dGTP and dCTP, and wherein the second set of nucleotides comprises a mixture of dGTP and dCTP, and is substantially free of dATP and dTTP. 
     
     
         16 . The system of  claim 13 , further comprising an exonuclease for cleaving linear molecular inversion probes and nucleic acid fragments following formation of the circularized probe compositions. 
     
     
         17 . The system of  claim 13 , further comprising a restriction enzyme or a uracil-N-glycosylase for cleaving the circularized probe compositions to form linearized probe compositions. 
     
     
         18 . The system of  claim 17 , further comprising primers and a polymerase configured to enable amplification of linearized probe compositions prior to contacting the linearized probe compositions to the capture device. 
     
     
         19 . The system of  claim 13 , further comprising a detector for detecting signals indicative of presence or absence of the first nucleotide variant and the second nucleotide variant. 
     
     
         20 . A system for analyzing a mixed nucleic acid sample obtained from an organism, comprising:
 a plurality of linear molecular inversion probes capable of hybridizing to a mixed nucleic acid population that includes a major subpopulation and a minor subpopulation, wherein the major and minor subpopulations each include a target sequence located in a first chromosomal region and containing a polymorphic site, wherein the polymorphic site includes combinations of a first nucleotide variant and a second nucleotide variant;   a ligase for forming circularized probe compositions from the linear molecular inversion probes;   an exonuclease for cleaving linear molecular inversion probes and nucleic acid fragments following formation of the circularized probe compositions;   a restriction enzyme or a uracil-N-glycosylase for cleaving the circularized probe compositions to form linearized probe compositions;   primers and a polymerase configured to enable amplification of linearized probe compositions prior to contacting the linearized probe compositions to the capture device;   a first set of nucleotides; and   a second set of nucleotides different from the first set of nucleotides.