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, i.e. the ligation of two or more fixed sequence oligonucleotides and one or more bridging oligonucleotides complementary to a region between the fixed sequence oligonucleotides.

We claim: 
     
         1 . A method for detecting nucleic acid regions of interest 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 set of fixed sequence oligonucleotides to specifically hybridize to complementary regions in the nucleic acid regions of interest;   introducing one or more bridging oligonucleotides to the genetic sample under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acid regions of interest, wherein the one or more bridging oligonucleotides are complementary to a region between the region of the nucleic acid region of interest complementary to the first and second fixed sequence oligonucleotides of a set, and wherein the one or more bridging oligonucleotides hybridize contiguously between the first and second fixed sequence oligonucleotides;   ligating the hybridized oligonucleotides to create a ligation product complementary to the nucleic acid region of interest;   amplifying the ligation product to create amplification products that reflect the relative frequency of the nucleic acid regions of interest in the genetic sample; and   detecting and quantifying the amplification products thereby detecting the nucleic acid regions of interest in the genetic sample.   
     
     
         2 . The method of  claim 1 , wherein one or both of the first or second fixed sequence oligonucleotides of the at least two sets of fixed sequence oligonucleotides comprise universal primer regions. 
     
     
         3 . The method of  claim 1 , wherein the first and second fixed sequence oligonucleotides are introduced prior to introduction of the bridging oligonucleotides. 
     
     
         4 . The method of  claim 1  wherein unhybridized fixed sequence oligonucleotides are removed prior to amplification of the contiguous ligation product. 
     
     
         5 . The method of  claim 3 , 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 1 , 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 the first or second fixed sequence oligonucleotide comprises one or more indices. 
     
     
         8 . The method of  claim 7 , wherein the first or second fixed sequence oligonucleotide comprises a locus index. 
     
     
         9 . The method of  claim 8 , wherein the amplification products are detected and quantified by hybridization of the locus index to an array. 
     
     
         10 . The method of  claim 8 , wherein each locus index is associated with a different label, and the amplification products are detected and quantified by the labels. 
     
     
         11 . The method of  claim 10 , wherein the label is fluorescent. 
     
     
         12 . The method of  claim 7 , wherein the one or more indices comprises an allele index and wherein a bridging oligonucleotide complementary for a specific polymorphism is used with a corresponding allele index. 
     
     
         13 . A method for detecting nucleic acid regions of interest 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 set of fixed sequence oligonucleotides to specifically hybridize to complementary regions in the nucleic acid regions of interest, and wherein at least one of the first and second fixed sequence oligonucleotide of each set comprises a locus index;   introducing one or more bridging oligonucleotides to the genetic sample under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acid regions of interest, wherein the one or more bridging oligonucleotides are complementary to a region between the region of the nucleic acid region of interest complementary to the first and second fixed sequence oligonucleotides of a set, and wherein the one or more bridging oligonucleotides hybridize contiguously between the first and second fixed sequence oligonucleotides;   ligating the hybridized oligonucleotides to create a ligation product complementary to the nucleic acid region of interest;   amplifying the ligation product to create amplification products that reflect the relative frequency of the nucleic acid regions of interest in the genetic sample; and   detecting and quantifying the amplification products thereby detecting the nucleic acid regions of interest in the genetic sample.   
     
     
         14 . The method of  claim 13 , wherein one or both of the first or second fixed sequence oligonucleotides of the at least two sets of fixed sequence oligonucleotides comprise universal primer regions. 
     
     
         15 . The method of  claim 13 , 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 13 , wherein the one or more bridging oligonucleotides are introduced simultaneously with the first and second fixed sequence oligonucleotides. 
     
     
         17 . The method of  claim 13 , wherein the amplification products are detected and quantified by hybridization of the locus index to an array. 
     
     
         18 . The method of  claim 13 , wherein each locus index is associated with a different label, and the amplification products are detected and quantified by the labels. 
     
     
         19 . The method of  claim 18 , wherein the label is fluorescent. 
     
     
         20 . The method of  claim 13 , wherein the at least one of the first and second fixed sequence oligonucleotides of each set comprises an allele index and wherein a bridging oligonucleotide complementary for a specific polymorphism is used in with a corresponding allele index.