LIGATION-BASED DETECTION OF GENETIC VARIANTS
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 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 sets of fixed sequence oligonucleotides to specifically hybridize to complementary regions in the nucleic acid regions of interest, wherein the first fixed sequence oligonucleotide of at least one set is specific for a polymorphism in a nucleic acid region 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 to detect nucleic acid regions of interest in a genetic sample.
2 . The method of claim 1 , wherein the first fixed sequence oligonucleotides specific for a polymorphism in a nucleic acid region of interest further comprise an allele index and wherein first fixed sequence oligonucleotides that are not specific for a polymorphism in a nucleic acid region of interest comprise a locus index.
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 3 , wherein the universal primer regions are used to amplify the ligation products and resulting amplification products are quantified by next generation sequencing of the allele and locus indices.
5 . The method of claim 3 , wherein the universal primer regions are used to amplify the ligation products and resulting amplification products are quantified by hybridization of the allele and locus indices to an array.
6 . The method of claim 2 , wherein each locus index and each allele index is associated with a different label, and the amplification products are detected and quantified by the labels.
7 . The method of claim 6 , wherein the labels are fluorescent.
8 . The method of claim 1 , wherein the first and second fixed sequence oligonucleotides are introduced prior to introduction of the bridging oligonucleotides.
9 . The method of claim 8 , 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.
10 . The method of claim 1 , wherein the one or more bridging oligonucleotides are introduced simultaneously with the first and second fixed sequence oligonucleotides.
11 . The method of claim 10 , wherein unhybridized fixed sequence oligonucleotides and bridging oligonucleotides are removed prior to amplification of the ligation product.
12 . 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 each nucleic acid region of interest, wherein the first fixed sequence oligonucleotides comprise a locus index and the first fixed sequence oligonucleotide of at least one set is specific for a polymorphism in a nucleic acid region 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 to detect nucleic acid regions of interest in a genetic sample.
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 13 , wherein the first fixed sequence oligonucleotides specific for a polymorphism in a nucleic acid region of interest further comprise an allele index.
15 . The method of claim 14 , wherein the universal primer regions are used to amplify the ligation products and resulting amplification products are quantified by next generation sequencing of the allele and locus indices.
16 . The method of claim 14 , wherein the universal primer regions are used to amplify the ligation products and resulting amplification products are quantified by hybridization of the allele and locus indices to an array.
17 . The method of claim 16 , wherein the resulting amplification products comprise fluorophores.
18 . The method of claim 12 , wherein the first and second fixed sequence oligonucleotides are introduced prior to introduction of the bridging oligonucleotides.
19 . The method of claim 18 , 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.
20 . The method of claim 12 , wherein the one or more bridging oligonucleotides are introduced simultaneously with the first and second fixed sequence oligonucleotides.
21 . The method of claim 20 , wherein unhybridized fixed sequence oligonucleotides and bridging oligonucleotides are removed prior to amplification of the ligation product.