Methods of enriching for and identifying polymorphisms

A method of identifying nucleic acid sequence differences with respect to a reference sequence comprising:

• a) cleaving a nucleic acid sample from one or more individuals with one or more sequence-specific cleavage agents to produce nucleic acid fragments;
• b) operatively linking said fragments with molecules capable of being replicated;
• c) introducing the linked molecules of step (b) into a system capable of replicating only a subset of said linked molecules, and replicating said subset to form a collection of replicated molecules;
• and
• d) detecting one or more nucleic acid sequence differences with respect to a reference sequence in the members of said collection of step (c) with a method capable of detecting one or more nucleotide differences with respect to a reference sequence.

A method of identifying nucleic acid sequence differences with respect to a reference sequence comprising:

• a) cleaving a nucleic acid sample from one or more individuals with one or more sequence-specific cleavage agents to produce nucleic acid fragments;
• b) operatively linking said subset of step (a) with said separation element;
• c) separating said linked molecules;
• and
• d) detecting one or more nucleic acid sequence differences with respect to a reference sequence in the members of said separated molecules of step (c) with a method capable of detecting one or more nucleotide differences with respect to a reference sequence.

A method of enriching for and identifying nucleic acid sequence differences with respect to a reference sequence comprising:

• a) fragmenting a nucleic acid sample from one or more individuals to an average fragment length;
• b) physically separating a subset of the nucleic acid fragments generated in step (a) based on the presence or absence of a particular nucleotide sequence within said fragments;
• c) operatively linking said subset of step (b) with molecules capable of being replicated;
• d) introducing the linked molecules of step (c) into a system capable of replicating said linked molecules, and replicating said linked molecules to form a collection of replicated molecules;
• and
• e) detecting a nucleic acid sequence difference with respect to a reference sequence in the collection of replicated molecules of step (d) using a method capable of detecting one or more nucleotide differences with respect to a reference sequence.

A method of enriching for and identifying nucleic acid sequence differences with respect to a reference sequence comprising:

• a) hybridizing a nucleic acid sample from one or more individuals with oligonucleotide primers under conditions wherein each of said primers permits extension by a polymerase at two-or more different sequences, and wherein the sequences replicated by extension of said primers comprise regions where there are known sequence differences between individuals of the species being examined;
• b) extending said oligonucleotide primers hybridized in step (a) to form an enriched collection of replicated molecules;
• and
• c) detecting one or more nucleic acid sequence differences in the members of said collection with respect to a reference sequence with a method capable of detecting one or more nucleotide differences with respect to a reference sequence.

A method of enriching for and identifying nucleic acid sequence differences with respect to a reference sequence comprising:

• a) fragmenting a nucleic acid sample from one or more individuals;
• b) physically separating a subset of said nucleic acid fragments based on the size of the fragments.
• c) operatively linking said subset of step (b) with molecules capable of being replicated;
• d) introducing the linked subset of molecules of step (c) into a system capable of replicating said linked subset of molecules, and replicating said subset of linked molecules to form an enriched collection of replicated molecules;
• and
• e) detecting one or more nucleotide sequence differences in the members of said collection of step (d) with a method capable of detecting one or more nucleotide differences with respect to a reference sequence.

A method for accessing a sub-population of a genome, such method comprising:

• a) cleaving a nucleic acid sample with a first restriction endonuclease wherein the recognition sequence of said first restriction endonuclease occurs infrequently in the genome;
• b) ligating an adapter molecule to the cleaved ends generated in step (a), said adapter having an overhang complementary to that generated by said first restriction endonuclease, and ligation of said adapter further regenerating all or part of the recognition sequence of said first restriction endonuclease;
• c) mixing an oligonucleotide primer complementary to said adapter molecule, wherein the 3′ terminus of said oligonucleotide primer is complementary to the regenerated recognition sequence of said first restriction endonuclease, under conditions which permit hybridization of said oligonucleotide primer to said adapter;
• and
• d) adding deoxynucleotides and a template-dependent DNA polymerizing activity under conditions which permit extension of said oligonucleotide primer, the resulting population of primer extension products comprising a sub-portion of the molecules in said nucleic acid sample.

A method for accessing a sub-population of a genome, such method comprising:

• a) cleaving a nucleic acid sample with one or more cleavage agents to produce nucleic acid fragments;
• b) operatively linking an adapter molecule to the cleaved ends generated in step (a);
• c) mixing a primer complementary to said adapter molecule with the linked molecules generated in step (b) under conditions that permit hybridization of said primer to said adapter;
• and
• d) incubating with a polymerizing activity under conditions that permit nucleic acid synthesis from said adapter, the resulting population of primer extension products comprising a sub-portion of said genome, wherein said sub-portion of said genome comprises an incomplete extension product.

A method for accessing a sub-population of a genome, such method comprising:

• a) cleaving a nucleic acid sample with a cleavage agent;
• b) operatively linking an adapter molecule to the cleaved ends generated in step (a), said adapter having an end compatible with that generated by said cleavage agent;
• c) mixing a primer complementary to said adapter molecule, wherein the 3′ terminus of said primer is complementary to the recognition sequence of said cleavage agent, under conditions that permit hybridization of said primer to said adapter;
• and
• d) adding deoxynucleotides and a template-dependent polymerizing activity under conditions that permit extension of said oligonucleotide primer, the resulting population of primer extension products comprising a sub-portion of said genome.