Methods of enriching for and identifying polymorphisms

• US 20070248979A1
• Filed: 04/11/2007
• Published: 10/25/2007
• Est. Priority Date: 06/23/1999
• Status: Active Grant

• Current Assignee: Catalyst Assets LLC
• Original Assignee: Stratagene California
• Inventors: Sorge, Joseph, Davis, Ronald
• Granted Patent: US 8,263,338 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/531 characterised by the capture moiety being a protein for target oligonucleotides

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.

Claims

1. A method of genotyping a single nucleotide polymorphism (SNP) in a DNA fragment of a subset population of DNA fragments comprising:

a) providing a human genomic DNA sample cleaved by a SpeI restriction endonuclease and comprising at least 10,000 DNA fragments;

b) contacting said DNA sample with a molecule comprising a sequence-specific binding activity under conditions which permit specific binding, wherein;

(i) said molecule comprising sequence-specific binding activity is a Gal4 transcription factor or DNA binding domain thereof; and

(ii) said DNA sample comprises a subset population of DNA fragments having a sequence that binds to said molecule comprising sequence-specific binding activity, and wherein a bound subset population of DNA fragments is retained by the molecule comprising sequence-specific binding activity, such that the subset population of bound DNA fragments is enriched for fragments comprising the sequence recognized by the molecule comprising sequence specific binding activity; and

c) detecting a sequence difference with respect to a reference sequence at a SNP site in a DNA fragment of the subset population of DNA fragments, wherein the presence or absence of a nucleotide sequence difference at a SNP site is detected in at least 10,000 primer extension products,

thereby genotyping a SNP in a DNA fragment of a subset population of DNA fragments.