Methods of enriching for and identifying polymorphisms

Abstract

The invention encompasses methods for enriching for and identifying a polymorphism within a nucleic acid sample either by separating a subset of a nucleic acid sample or by selectively replicating a subset of a nucleic acid sample such that the polymorphism is contained within a nucleic acid population with reduced complexity, and then identifying the polymorphism within the enriched nucleic acid sample. Methods also are disclosed for enriching for and identifying a polymorphism by contacting a nucleic acid sample that includes a subset of nucleic acid molecules having a sequence that binds to a sequence-specific binding activity with a molecule having a sequence-specific binding activity under conditions which permit specific binding, such that the subset of nucleic acid molecules bound to the activity is enriched for nucleic acid molecules having the sequence recognized by the sequence-specific binding activity, and detecting a polymorphism with respect to a reference sequence in the subset of nucleic acid molecules.

1 . (canceled) 
     
     
         2 . A method of enriching for and identifying the presence or absence of nucleic acid sequence differences with respect to a reference sequence, the method comprising:
 a) providing one or more enriched subsets of a nucleic acid sample and/or a complement of said subsets, comprising:   1) fragmenting a nucleic acid sample to yield an average fragment length; and   2) physically separating one or more subsets of the nucleic acid fragments generated after fragmenting by binding the fragments to one or more solid supports based on the presence or absence of one or more particular nucleotide sequences within the fragments, such that at least 10,000 non-identical nucleic acid fragments are bound to the solid supports; and   3) amplifying the fragments bound to the solid supports to produce enriched subsets, wherein said enriched subsets are a fraction of the complexity of the un-enriched nucleic acid sample;   b) determining in the at least 10,000 non-identical fragments of said enriched subsets the nucleotides present at polymorphic sites.   
     
     
         3 . The method of  claim 2  wherein determining the nucleotides present at polymorphic sites comprises hybridizing sequencing primers to the bound and amplified fragments. 
     
     
         4 . The method of  claim 2  wherein the polymorphic sites are single nucleotide polymorphism (SNP) sites and the nucleotides present at said SNP sites are determined in each of at least 10,000 non-identical fragments of said enriched subsets. 
     
     
         5 . The method of  claim 2  wherein the particular nucleotide sequence within the fragments is an adapter sequence. 
     
     
         6 . The method of  claim 5  wherein the adapter sequence is linked to the fragment. 
     
     
         7 . The method of  claim 5  wherein the adapter sequence is operatively linked to the fragment. 
     
     
         8 . The method of  claim 5  wherein the adapter sequence is ligated to the fragment. 
     
     
         9 . The method of  claim 2  wherein amplifying the fragments and determining the nucleotides present at polymorphic sites occurs on the same solid support. 
     
     
         10 . The method of  claim 2  wherein two nucleic acid primers are utilized in amplifying the solid support bound fragments and each said primer is complementary to a different sequence within the fragments. 
     
     
         11 . A method of enriching for and identifying the presence or absence of nucleic acid sequence differences with respect to another sequence, the method comprising:
 a) providing one or more enriched subsets of a nucleic acid sample and/or one or more complements of said subsets, wherein said enriched subsets are produced by a method comprising: fragmenting a nucleic acid sample to an average fragment length, and physically separating one or more subsets of the nucleic acid fragments generated by fragmenting by hybridizing the fragments to oligonucleotides bound a solid support based on the presence or absence of a particular nucleotide sequence within the fragments, such that said separated one or more enriched subsets is separated from the nucleic acid sample, wherein said providing step (a) comprises:   1) said one or more enriched subsets contains at least one anchor sequence present at a rate selected from the group consisting of at least 50, at least 100, at least 1000, at least 10,000, at least 25,000, at least 50,000 and at least 100,000 times within the fragments;   2) amplifying the fragments with a primer attached to the solid support that binds to said anchor sequence;   3) said one or more enriched subsets are a fraction of the complexity of the un-enriched nucleic acid sample; and   b) determining the nucleotides present at polymorphic sites in non-identical anchor sequence-containing fragments of said one or more enriched subsets and/or complements of said enriched subsets.   
     
     
         12 . The method of  claim 11  wherein the polymorphic sites are single nucleotide polymorphism (SNP) sites and the nucleotides present at said SNP sites are determined in each of at least 10,000 non-identical fragments of said enriched subsets. 
     
     
         13 . The method of  claim 11 , wherein said primer is labeled with a moiety which permits attachment of said primer to a solid support. 
     
     
         14 . The method of  claim 11  wherein the particular nucleotide sequence within the fragments is an adapter sequence. 
     
     
         15 . The method of  claim 13  wherein said primer binds to said particular nucleotide sequence present within said fragments. 
     
     
         16 . The method of  claim 14  wherein the adapter sequence is operatively linked to the fragment. 
     
     
         17 . The method of  claim 14  wherein the adapter sequence is ligated to the fragment. 
     
     
         18 . The method of  claim 11  wherein determining the nucleotides present at polymorphic sites comprises hybridizing sequencing primers to the bound and amplified fragments. 
     
     
         19 . The method of  claim 11  wherein amplifying the fragments and determining the nucleotides present at polymorphic sites occurs on the same solid support. 
     
     
         20 . The method of  claim 11  wherein two nucleic acid primers are utilized in amplifying the solid support bound fragments and each said primer is complementary to a particular nucleotide sequence within the fragments. 
     
     
         21 . A method of enriching for and identifying the presence or absence of nucleic acid sequence differences with respect to a reference sequence, the method comprising:
 a) providing one or more enriched subsets of a nucleic acid sample and/or complements thereof, by a method comprising: fragmenting a nucleic acid sample to an average fragment length and physically separating one or more subsets of the nucleic acid fragments generated by fragmenting based on the presence or absence of a particular nucleotide sequence within the fragments such that said one or more enriched subsets is separated from the nucleic acid sample, wherein:   1) said enriched subsets comprise non-identical nucleic acid fragments and are a fraction of the complexity of the un-enriched nucleic acid sample; and   2) said enriched subsets are replicated by a method comprising replication with a nucleic acid primer which is bound to a solid support and is complementary to a selected sequence of said enriched subsets under conditions wherein said primer permits nucleic acid extension and possesses at least one 3′ terminal selective nucleotide; and   b) determining the nucleotides present at polymorphic sites in the non-identical nucleic acid fragments and/or complements of said nucleic acid fragments.   
     
     
         22 . The method of  claim 21 , wherein said primer is labeled with a moiety which permits attachment of said primer to a solid support. 
     
     
         23 . The method of  claim 21  wherein the particular nucleotide sequence within the fragments is an adapter sequence. 
     
     
         24 . The method of  claim 22  wherein said primer binds to said particular nucleotide sequence present within said fragments. 
     
     
         25 . The method of  claim 23  wherein the adapter sequence is operatively linked to the fragment. 
     
     
         26 . The method of  claim 23  wherein the adapter sequence is ligated to the fragment. 
     
     
         27 . The method of  claim 21  wherein the polymorphic sites are single nucleotide polymorphism (SNP) sites and the nucleotides present at said SNP sites are determined in each of at least 10,000 non-identical fragments of said enriched subsets. 
     
     
         28 . The method of  claim 21  wherein amplifying the fragments and determining the nucleotides present at polymorphic sites occurs on the same solid support. 
     
     
         29 . The method of  claim 21  wherein two nucleic acid primers are utilized in amplifying the solid support bound fragments and each said primer is complementary to a particular nucleotide sequence within the fragments.