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.

1 . A set of oligonucleotides for ligation-based detection of a nucleic acid region of interest, comprising:
 a first oligonucleotide that comprises sequences complementary to the sequences of a first portion of a nucleic acid region, and a universal primer sequence;   a second oligonucleotide that comprises sequences complementary to the sequence of a second portion of a nucleic acid region and a universal primer sequence; and   one or more bridging oligonucleotides that are complementary to the region immediately adjacent and between the nucleic acid region complementary to the first and second oligonucleotides.   
     
     
         2 . The set of oligonucleotides of  claim 1 , wherein the set comprises two or more bridging oligonucleotides with the ability to identify different polymorphisms within the nucleic acid of interest. 
     
     
         3 . The set of oligonucleotides of  claim 1 , wherein the bridging molecules provide degeneracy for one or more internal position of the bridging oligonucleotide. 
     
     
         4 . The set of oligonucleotides of  claim 1 , wherein the first oligonucleotide further comprises one or more indices. 
     
     
         5 . The set of oligonucleotides of  claim 1 , wherein the second oligonucleotide further comprises one or more indices. 
     
     
         6 . The set of oligonucleotides of  claim 4 , wherein the indices include a sample index. 
     
     
         7 . The set of oligonucleotides of  claim 5 , wherein the indices include a sample index. 
     
     
         8 . The set of oligonucleotides of  claim 4 , wherein the indices include a locus index. 
     
     
         9 . The set of oligonucleotides of  claim 5 , wherein the indices include a locus index. 
     
     
         10 . The set of oligonucleotides of  claim 4 , wherein the indices include an allele index. 
     
     
         11 . The set of oligonucleotides of  claim 5 , wherein the indices include an allele index. 
     
     
         12 . An assay system for detecting a nucleic acid region of interest in a genetic sample, comprising the steps of:
 providing a genetic sample;   introducing a first and second fixed sequence oligonucleotide to the genetic sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions in the nucleic acid of interest;   introducing one or more bridging oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acid of interest, wherein the one or more bridging oligonucleotides are complementary to a region of the nucleic acid between and immediately adjacent to the region complementary to the first and second fixed sequence oligonucleotides;   ligating the hybridized oligonucleotides to create a contiguous ligation product complementary to the nucleic acid region of interest;   amplifying the contiguous ligation product to create amplification products having the sequence of the nucleic acid region; and   detecting and quantifying the amplification products;   wherein detection of the amplification product provides detection of the nucleic acid region in the genetic sample.   
     
     
         13 . The assay system of  claim 12 , wherein the fixed sequence oligonucleotides comprise universal primer regions that are used in amplification of the contiguous ligation product. 
     
     
         14 . The assay system of  claim 12 , wherein the unhybridized fixed sequence oligonucleotides are removed prior to amplification of the contiguous ligation product. 
     
     
         15 . The assay system of  claim 12 , wherein the first and second fixed sequence oligonucleotides are introduced prior to introduction of the bridging oligonucleotides. 
     
     
         16 . The assay system of  claim 12 , wherein the hybridization products of the fixed sequence oligonucleotides and the nucleic acid region are isolated prior to introduction of the bridging oligonucleotides. 
     
     
         17 . The assay system of  claim 12 , wherein the one or more bridged oligonucleotides are introduced simultaneously with the first and second fixed sequence oligonucleotides. 
     
     
         18 . The assay system of  claim 12 , wherein the amplification products are optionally isolated and quantified. 
     
     
         19 . The assay system of  claim 12 , wherein the first or second oligonucleotide comprises one or more indices. 
     
     
         20 . The assay system of  claim 19 , wherein the amplification product is detected by detection of the one or more indices. 
     
     
         21 . The assay system of  claim 19 , wherein the first or second fixed sequence oligonucleotide comprises an allele index, and wherein a bridging oligonucleotide complementary for a specific polymorphism is used in the hybridization with the corresponding allele index. 
     
     
         22 . The assay system of  claim 12 , wherein the first or second fixed sequence oligonucleotides are allele-specific. 
     
     
         23 . The assay system of  claim 12 , wherein the one or more bridging oligonucleotides are allele-specific. 
     
     
         24 . An assay system for detecting a nucleic acid region of interest in a maternal sample, comprising the steps of:
 providing a maternal sample comprising cell free DNA from both maternal and fetal sources;   introducing a first and second fixed sequence oligonucleotide to the genetic sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions in the nucleic acid of interest;   introducing one or more bridging oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acid of interest, wherein one or more bridging oligonucleotides are complementary to a region of the nucleic acid between and immediately adjacent to the region complementary to the first and second fixed sequence oligonucleotides;   ligating the hybridized oligonucleotides to create a contiguous ligation product complementary to the nucleic acid region of interest; and   amplifying the contiguous ligation product to create amplification products having the sequence of the nucleic acid region; and   detecting and quantifying the amplification products;   wherein quantification of the amplification product provides a relative frequency of the nucleic acid region in the maternal sample.   
     
     
         25 . The assay system of  claim 24 , wherein the fixed sequence oligonucleotides comprise universal primer regions that are used in amplification of the contiguous ligation product. 
     
     
         26 . The assay system of  claim 24 , wherein the unhybridized fixed sequence oligonucleotides are removed prior to amplification of the contiguous ligation product. 
     
     
         27 . The assay system of  claim 24 , wherein the first and second fixed sequence oligonucleotides are introduced prior to introduction of the bridging oligonucleotides. 
     
     
         28 . The assay system of  claim 24 , wherein the hybridization products of the fixed sequence oligonucleotides and the nucleic acid region are isolated prior to introduction of the bridging oligonucleotides. 
     
     
         29 . The assay system of  claim 24 , wherein the one or more bridged oligonucleotides are introduced simultaneously with the first and second fixed sequence oligonucleotides. 
     
     
         30 . The assay system of  claim 24 , wherein the amplification products are optionally isolated and quantified. 
     
     
         31 . The assay system of  claim 24 , wherein the first or second oligonucleotide comprises one or more indices. 
     
     
         32 . The assay system of  claim 31 , wherein the amplification products are detected and quantified by the detection of the one or more indices. 
     
     
         33 . The assay system of  claim 31 , wherein the first or second oligonucleotide comprises an allele index, and wherein a bridging oligonucleotide complementary for a specific polymorphism is used in the hybridization with the corresponding allele index. 
     
     
         34 . A set of oligonucleotides for extension and ligation-based detection of a nucleic acid region of interest, comprising:
 a first oligonucleotide that comprises sequences complementary to the sequences of a first portion of a nucleic acid region, and a universal primer sequence;   a second oligonucleotide that comprises sequences complementary to the sequence of a second portion of a nucleic acid region and a universal primer sequence; and   one or more bridging oligonucleotides that are complementary to the region between the nucleic acid region complementary to the first and second oligonucleotides, wherein a gap of one base or more exists between a bridging oligonucleotide and the first and/or second fixed sequence oligonucleotides.   
     
     
         35 . An assay system for detecting a nucleic acid region of interest in a genetic sample, comprising the steps of:
 providing a genetic sample;   introducing a first and second fixed sequence oligonucleotide to the genetic sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions in the nucleic acid of interest;   introducing one or more bridging oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acid of interest, wherein the one or more bridging oligonucleotides are complementary to a region of the nucleic acid between the first and second fixed sequence oligonucleotides, wherein a gap of one base or more exists between a bridging oligonucleotide and the first and/or second fixed sequence oligonucleotides;   extending one or more of the hybridized oligonucleotides to create contiguous hybridized oligonucleotides;   ligating the contiguous hybridized oligonucleotides to create a contiguous ligation product complementary to the nucleic acid region of interest;   amplifying the contiguous ligation product to create amplification products having the sequence of the nucleic acid region; and   detecting and quantifying the amplification products;   wherein detection of the amplification product provides detection of the nucleic acid region in the genetic sample.   
     
     
         36 . The assay system of  claim 35 , wherein the one or more oligonucleotides are extended by addition of dNTPs and a polymerase. 
     
     
         37 . The assay system of  claim 36 , wherein the fixed sequence oligonucleotides comprise universal primer regions that are used in amplification of the contiguous ligation product. 
     
     
         38 . The assay system of  claim 35 , wherein the unhybridized fixed sequence oligonucleotides are removed prior to amplification of the contiguous ligation product. 
     
     
         39 . The assay system of  claim 35 , wherein the first and second fixed sequence oligonucleotides are introduced prior to introduction of the bridging oligonucleotides. 
     
     
         40 . The assay system of  claim 35 , wherein the hybridization products of the fixed sequence oligonucleotides and the nucleic acid region are isolated prior to introduction of the bridging oligonucleotides. 
     
     
         41 . The assay system of  claim 35 , wherein the one or more bridged oligonucleotides are introduced simultaneously with the first and second fixed sequence oligonucleotides. 
     
     
         42 . The assay system of  claim 35 , wherein the amplification products are optionally isolated and quantified. 
     
     
         43 . The assay system of  claim 35 , wherein the first or second oligonucleotide comprises one or more indices. 
     
     
         44 . The assay system of  claim 35 , wherein the amplification product is detected by detection of the one or more indices. 
     
     
         45 . The assay system of  claim 44 , wherein the first or second fixed sequence oligonucleotide comprises an allele index, and wherein a bridging oligonucleotide complementary for a specific polymorphism is used in the hybridization with the corresponding allele index. 
     
     
         46 . The assay system of  claim 35 , wherein the first or second fixed sequence oligonucleotides are allele-specific.