Ligation-based detection of genetic variants

Abstract

The present invention provides assays systems and methods for detection of genetic variants in a sample, including copy number variation and single nucleotide polymorphisms. The invention preferably employs the technique of tandem ligation—e.g., the ligation of two or more fixed sequence oligonucleotides and one or more bridging oligonucleotides complementary to a region between the fixed sequence oligonucleotides—combined with detection of levels of particular genomic regions using array hybridization.

We claim: 
     
         1 . A method for detecting a variance in the frequency of a genomic region in a genetic sample, comprising the steps of:
 providing a genetic sample;   introducing at least two sets of first and second fixed sequence oligonucleotides to the genetic sample under conditions that allow the sets of fixed sequence oligonucleotides to specifically hybridize to complementary regions in nucleic acid regions of interest, wherein each set comprises an oligonucleotide associated with an optically detectable label, and wherein both sets comprise a region that binds selectively to a single array feature;   ligating the hybridized oligonucleotides to create contiguous ligation products complementary to nucleic acid regions of interest;   introducing the contiguous ligation products from both sets to an array comprising one or more features complementary to the contiguous ligation products; and   detecting hybridization of the contiguous ligation products from the first and second set to the array by detection of the optically detectable labels;   wherein the relative frequency of the optically detectable labels on the array is indicative of the presence or absence of a variance in the frequency of a nucleic acid region of interest in the genetic sample.   
     
     
         2 . The method of  claim 1 , further comprising amplifying the contiguous ligation products prior to introduction to the array. 
     
     
         3 . The method of  claim 2 , wherein one or both of the first or second fixed sequence oligonucleotides of each set comprise universal primer regions. 
     
     
         4 . The method of  claim 1 , further comprising introducing one or more bridging oligonucleotides for each set under conditions that allow the bridging oligonucleotides to hybridize to complementary regions in the nucleic acid regions of interest between the first and second fixed sequence oligonucleotides to create hybridization products. 
     
     
         5 . The method of  claim 4 , wherein the hybridization products of the fixed sequence oligonucleotides and the nucleic acid regions of interest to which they hybridize are isolated prior to introduction of the bridging oligonucleotides. 
     
     
         6 . The method of  claim 4 , wherein the one or more bridging oligonucleotides are introduced simultaneously with the first and second fixed sequence oligonucleotides. 
     
     
         7 . The method of  claim 1 , wherein hybridization of the contiguous ligation products comprises hybridization to individual oligonucleotides bound to the array. 
     
     
         8 . The method of  claim 1 , wherein the label is a fluorescent label. 
     
     
         9 . The method of  claim 1 , wherein the variance is detected by a variation from the expected ratio of nucleic acids of interest in the genetic sample. 
     
     
         10 . The method of  claim 9 , wherein the variance is detected by an increased or decreased level of hybridization of one set of contiguous ligation products as compared to the second set of contiguous ligation products. 
     
     
         11 . A method for detecting regions of interest corresponding to a first and second chromosome in a genetic sample, comprising the steps of:
 providing a genetic sample;   introducing at least two sets of first and second fixed sequence oligonucleotides to the genetic sample under conditions that allow the sets of fixed sequence oligonucleotides to specifically hybridize to complementary regions in nucleic acid regions of interest, wherein the first set of fixed sequence oligonucleotides is complementary to a genomic region on a first chromosome and the second set of fixed sequence oligonucleotides is complementary to a genomic region on a second chromosome, and wherein each set comprises an oligonucleotide associated with an optically detectable label, and wherein both sets comprise a region that binds selectively to a single array feature;   ligating the hybridized oligonucleotides to create contiguous ligation products complementary to nucleic acid regions of interest;   introducing the contiguous ligation products from both sets to an array comprising one or more features complementary to the contiguous ligation products; and   detecting hybridization of the contiguous ligation products from the first and second set to the array by detection of the optically detectable labels;   wherein the relative frequency of the optically detectable labels on the array is indicative of the presence or absence of a variance in the frequency of a first and second chromosome in the genetic sample.   
     
     
         12 . The method of  claim 11 , further comprising amplifying the contiguous ligation products prior to introduction to the array. 
     
     
         13 . The method of  claim 12 , wherein one or both of the first or second fixed sequence oligonucleotides of each set comprise universal primer regions. 
     
     
         14 . The method of  claim 11 , further comprising introducing one or more bridging oligonucleotides for each set under conditions that allow to bridging oligonucleotides to hybridize to complementary regions in the nucleic acid regions of interest between the first and second fixed sequence oligonucleotides. 
     
     
         15 . The method of  claim 14 , wherein the hybridization products of the fixed sequence oligonucleotides and the nucleic acid regions of interest to which they hybridize are isolated prior to introduction of the bridging oligonucleotides. 
     
     
         16 . The method of  claim 14 , wherein the one or more bridging oligonucleotides are introduced simultaneously with the first and second fixed sequence oligonucleotides. 
     
     
         17 . The method of  claim 11 , wherein hybridization of the contiguous ligation products comprises hybridization to individual oligonucleotides bound to the array. 
     
     
         18 . The method of  claim 11 , wherein the label is a fluorescent label. 
     
     
         19 . The method of  claim 11 , wherein the variance is detected by a variation from the expected ratio of nucleic acids of interest in the genetic sample. 
     
     
         20 . The method of  claim 19 , wherein the variance is detected by an increased or decreased level of hybridization of one set of contiguous ligation products as compared to the second set of contiguous ligation products. 
     
     
         21 . The method of  claim 11 , wherein the method is carried out for at least ten sets of fixed sequence oligonucleotides complementary to a genomic region on a first chromosome and at least ten sets of fixed sequence oligonucleotides complementary to a genomic region on a second chromosome. 
     
     
         22 . The method of  claim 21 , wherein the method is carried out for at least 100 sets of fixed sequence oligonucleotides complementary to a genomic region on a first chromosome and at least 100 sets of fixed sequence oligonucleotides complementary to a genomic region on a second chromosome. 
     
     
         23 . The method of  claim 22 , wherein the method is carried out for at least 200 sets of fixed sequence oligonucleotides complementary to a genomic region on a first chromosome and at least 200 sets of fixed sequence oligonucleotides complementary to a genomic region on a second chromosome. 
     
     
         24 . The method of  claim 23 , wherein the method is carried out for at least 500 sets of fixed sequence oligonucleotides complementary to a genomic region on a first chromosome and at least 500 sets of fixed sequence oligonucleotides complementary to a genomic region on a second chromosome.