Detection of Target Nucleic Acids using Hybridization

Abstract

The present invention provides detection systems and methods for detection of loci and genomic regions in a sample, including mixed samples, using hybridization to an array.

What is claimed is: 
     
         1 . An assay method for providing a statistical likelihood of a fetal copy number variation comprising:
 providing a maternal sample comprising maternal and fetal cell free DNA;   interrogating one or more loci from a first target genomic region using fixed sequence oligonucleotides comprising a region complementary to a locus in the first target genomic region and a capture region;   interrogating one or more loci from a second target genomic region using fixed sequence oligonucleotides comprising a region complementary to a locus in the second target genomic region and a capture region;   detecting the interrogated loci from the first and second target genomic region via hybridization of the capture regions of the fixed sequence oligonucleotides to an array comprising capture probes complementary to the capture regions;   quantifying the capture regions of the synthetic oligonucleotides to determine a relative frequency of the interrogated loci;   estimating the relative frequency of the first and second target genomic regions based on the quantified interrogated loci; and   calculating a statistical likelihood of a fetal copy number variation using the estimated relative quantity of the first and second target genomic regions in the sample.   
     
     
         2 . The assay method of  claim 1 , wherein the interrogation of the loci comprises hybridization of two or more fixed sequence oligonucleotides per locus, and wherein one of the fixed sequence oligonucleotides complementary to a locus comprises a capture region. 
     
     
         3 . The assay method of  claim 2 , wherein the fixed sequence oligonucleotides are ligated following hybridization. 
     
     
         4 . The assay method of  claim 3 , wherein an amplification step is performed after the hybridization and ligation. 
     
     
         5 . The assay method of  claim 4 , wherein the amplification is universal amplification using the polymerase chain reaction. 
     
     
         6 . The assay method of  claim 1 , wherein the capture region of one or more loci from the first genomic region and the capture region of one or more loci from the second genomic region are substantially the same, and wherein the capture regions compete for hybridization to the same capture probe. 
     
     
         7 . The assay method of  claim 1 , further providing:
 interrogating polymorphic loci from at least one target genomic region using allele-specific oligonucleotides;   detecting the alleles of the polymorphic loci;   quantifying the alleles of the polymorphic loci to estimate the fraction of fetal DNA in the sample; and   calculating a statistical likelihood of a fetal copy number variation in the maternal sample, wherein the relative frequency of loci from the first target genomic region, the relative frequency of loci from the second target genomic region, and the fraction of fetal DNA is used to provide a statistical likelihood of the presence of a fetal copy number variation.   
     
     
         8 . The method of  claim 7 , further providing:
 identifying low frequency alleles from the polymorphic loci where the maternal DNA is homozygous and the non-maternal DNA is heterozygous; and   using the low frequency alleles from the polymorphic loci to compute the fraction of fetal DNA.   
     
     
         9 . The method of  claim 1 , wherein the first genomic region is a chromosome. 
     
     
         10 . The method of  claim 1 , wherein the second genomic region is a chromosome. 
     
     
         11 . The method  claim 1 , wherein the first genomic region is a sub-chromosomal region. 
     
     
         12 . The method  claim 1 , wherein the second genomic region is a sub-chromosomal region. 
     
     
         13 . A method for determining a likelihood of a fetal aneuploidy comprising the steps of:
 providing a maternal sample comprising maternal and fetal cell free DNA;   introducing a first set of two or more fixed sequence oligonucleotides complementary to a locus in a first target genomic region in the maternal sample under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to the locus, wherein at least one of the fixed sequence oligonucleotides of each set comprises a universal primer site and a capture region;   introducing a second set of two or more fixed sequence oligonucleotides complementary to a locus in a second target genomic region in the maternal sample under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to the locus, wherein at least one of the fixed sequence oligonucleotides of each set comprises a universal primer site and a capture region;   introducing bridging oligonucleotides to the maternal sample under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the loci between the hybridized fixed sequence oligonucleotides;   ligating the hybridized first and second fixed sequence oligonucleotides and the bridging oligonucleotides to create ligation products corresponding to the loci;   amplifying the ligation products using the universal primer sites;   applying the capture regions of the amplified ligation products to an array, wherein the array comprises capture probes complementary to the capture regions;   quantifying a relative frequency of ligation products corresponding to loci from the first target genomic region and a relative frequency of ligation products corresponding to loci from the second target genomic region; and   computing a likelihood of a fetal aneuploidy using the relative frequency of ligation products corresponding to loci from the first and second target genomic regions to determine the likelihood of a fetal aneuploidy.   
     
     
         14 . The method of  claim 13  wherein the relative frequencies of the ligation products corresponding to the loci from the first and second target genomic region are combined and compared to calculate a target genomic region ratio. 
     
     
         15 . The method of  claim 13 , wherein the sample DNA is attached to a solid support prior to hybridization of the fixed sequence oligonucleotides. 
     
     
         16 . The method of  claim 13 , wherein the hybridization complexes are attached to a solid support following hybridization of the fixed sequence oligonucleotides. 
     
     
         17 . The method of  claim 13 , wherein the unhybridized oligonucleotides are removed following hybridization and/or ligation. 
     
     
         18 . The method of  claim 13 , wherein the entire amplification product is applied to the array. 
     
     
         19 . The method of  claim 13 , wherein the amplification product is cleaved following amplification, and wherein the portion comprising the capture region is applied to the array. 
     
     
         20 . The method of  claim 13 , wherein the entire amplification product is applied to the array. 
     
     
         21 . A method for determining a likelihood of a fetal aneuploidy comprising the steps of:
 providing a maternal sample comprising maternal and fetal cell free DNA;   introducing a first set of two or more fixed sequence oligonucleotides complementary to a locus in a first target genomic region in the maternal sample under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to the locus, wherein at least one of the fixed sequence oligonucleotides of each set comprises a universal primer site and a capture region;   introducing a second set of two or more fixed sequence oligonucleotides complementary to loci in a second target genomic region in the maternal sample under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to the loci, wherein at least one of the fixed sequence oligonucleotides of each set comprises a universal primer site and a capture region;   introducing third sets of two or more fixed sequence oligonucleotides complementary to a set of polymorphic loci in the maternal sample under conditions that allow a complementary region of each fixed sequence oligonucleotide to specifically hybridize to polymorphic loci, wherein at least one of the two fixed sequence oligonucleotides of each set comprises a universal primer site and a capture region;   introducing bridging oligonucleotides to the maternal sample under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the loci between the fixed sequence oligonucleotides;   ligating the hybridized first and second fixed sequence oligonucleotides and the bridging oligonucleotides to create ligation products corresponding to the loci;   amplifying the ligation products using the universal primer sites;   applying the capture region of the amplified ligation products to an array, wherein the array comprises capture probes complementary to the capture regions;   quantifying a relative frequency of each allele from the polymorphic loci to determine a percent fetal cell-free DNA in the sample;   quantifying a relative frequency of ligation products corresponding to loci from the first target genomic region and a relative frequency of ligation products corresponding to loci from the second target genomic region; and   computing a likelihood of a fetal aneuploidy using the relative frequency of ligation products corresponding to loci from the first and second target genomic regions and the percent fetal cell-free DNA to determine the likelihood of a fetal aneuploidy.   
     
     
         22 . The method of  claim 21 , further comprising comparing the relative frequencies of the ligation products corresponding to loci from the first and second target genomic regions and adjusting the likelihood of a fetal aneuploidy based on the percent fetal cell-free DNA. 
     
     
         23 . The method of  claim 21 , wherein the relative frequencies of the ligation products corresponding to the loci from the first and second target genomic region are combined and compared to calculate a target genomic region ratio. 
     
     
         24 . The method of  claim 21 , wherein the percent fetal cell free DNA is calculated by detecting levels of one or more non-maternal alleles. 
     
     
         25 . The method of  claim 24 , wherein the non-maternal alleles are autosomal loci. 
     
     
         26 . The method of  claim 24 , wherein the non-maternal alleles comprise one or more genetic variations compared to maternal alleles. 
     
     
         27 . The method of  claim 21 , wherein the ligation products from loci of the first and second target genomic regions and the ligation products from the polymorphic loci are amplified in a single vessel. 
     
     
         28 . The method of  claim 21 , wherein the entire amplification product is applied to the array. 
     
     
         29 . The method of  claim 21 , wherein the unhybridized oligonucleotides are removed following hybridization and/or ligation. 
     
     
         30 . The method of  claim 21 , wherein the amplification product is cleaved following amplification, and wherein the portion comprising the capture region is applied to the array.