Methods for genotyping selected polymorphism

Abstract

Methods for genotyping polymorphisms using a locus specific primer that is complementary to a region near a selected polymorphism are described. Methods for synthesizing pools of locus specific primers that incorporate some degenerate positions are also disclosed. A plurality of different sequence capture probes are synthesized simultaneously using degenerate oligonucleotide synthesis. The sequence of the locus specific regions of the capture probes are related in that they have some bases that are identical in each sequence in the plurality of sequences and positions that vary from one locus specific region to another. The sequences are selected based on proximity to a polymorphism of interest and because they conform to a similar sequence pattern.

1 .- 41 . (canceled) 
     
     
         42 . A kit for amplifying one or more target sequences from a nucleic acid sample, the kit comprising:
 one or more adaptors to be ligated to the target sequences to produce adaptor-ligated sequences, wherein the adaptors comprise a first priming sequence; and   one or more probes, wherein each probe comprises a second priming sequence and a target-specific region, and the probe can hybridize to the target sequence, but not the adaptor.   
     
     
         43 . The kit of  claim 42 , wherein the kit further comprises a reagent that fragmentizes the nucleic acid sample into a plurality of fragments such that the adaptors are ligated to the fragments. 
     
     
         44 . The kit of  claim 42 , wherein the kit further comprises a reagent that extends at least some of the probes using the adaptor-ligated sequences as templates such that an extended probe comprises a sequence that is complementary to the target sequence, a sequence that is complementary to the first priming sequence and the second priming sequence. 
     
     
         45 . The kit of  claim 44 , wherein the kit further comprises a reagent that amplifies the extended probe with primers to the first and second priming sequences or sequences complementary to the first and second priming sequences. 
     
     
         46 . The kit of  claim 45 , wherein the kit further comprises the primers to the first and second priming sequences or the sequences complementary to the first and second priming sequences. 
     
     
         47 . The kit of  claim 42 , wherein the probes are free in solution. 
     
     
         48 . The kit of  claim 42 , wherein the probes are attached to a solid support selected from the group consisting of arrays, beads, microparticles, microtitre dishes, and gels. 
     
     
         49 . The kit of  claim 48 , wherein the probes are attached to the solid support through a covalent interaction. 
     
     
         50 . The kit of  claim 49 , wherein each species of probes is attached to the solid support in a discrete location. 
     
     
         51 . The kit of  claim 49 , wherein each probe further comprises a tag sequence that is unique for each species of probes and the probes are attached to the solid support by hybridization to a collection of tag probes that are covalently attached to the solid support. 
     
     
         52 . The kit of  claim 44 , wherein the kit further comprises one or more labeled nucleotides to be incorporated into the extended probe, thereby generating a labeled extended probe. 
     
     
         53 . The kit of  claim 52 , wherein the labeled nucleotides comprise biotin. 
     
     
         54 . The kit of  claim 52 , wherein the labeled nucleotides comprise one or more selected from the group consisting of digoxigenin, Cyanine-3, Cyanine-5, Rhodamine, Texas Red and an antibody. 
     
     
         55 . The kit of  claim 44 , wherein the kit further comprises a reagent that digests a single stranded, non-extended probe. 
     
     
         56 . The kit of  claim 42 , wherein there are 2 or more different probes. 
     
     
         57 . The kit of  claim 42 , wherein there are 100 or more different probes. 
     
     
         58 . The kit of  claim 42 , wherein the probes have at least one degenerate base. 
     
     
         59 . The kit of  claim 42 , wherein the probes have between 4 to 10 degenerate bases.