Complexity Management of Genomic DNA

• US 20100292097A1
• Filed: 06/24/2010
• Published: 11/18/2010
• Est. Priority Date: 10/27/1999
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Dong, Shoulian
• Granted Patent: US 7,993,883 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The presently claimed invention provides for novel methods and kits for reducing the complexity of a nucleic acid sample by providing non-gel based methods for amplification of a subset of the sequences in a sample. In a preferred embodiment, amplification of a subset can be accomplished by digesting a sample with two or more restriction enzymes and ligating adaptors to the fragments so that only a subset of the fragments can be amplified. The invention further provides for analysis of the above amplified sample by hybridization to an array, which may be specifically designed to interrogate the desired fragments for particular characteristics, such as, for example, the presence or absence of a polymorphism.

Claims

1-34. -34. (canceled)

35. A method of determining the base present at a nucleotide polymorphism in a population of individuals comprising:

providing a first nucleic acid sample from each of the individuals;

providing a second nucleic acid samples by;

fragmenting the first nucleic acid sample using a first and a second restriction enzyme to produce fragments wherein the polymorphism is predicted to be on a fragment that is cut on one end by the first restriction enzyme and on the other end by the second restriction enzyme; and

wherein the polymorphism is a single nucleotide polymorphism (SNP);

ligating a first and a second adaptor to the fragments to produce adaptor-ligated fragments, wherein the first adaptor has a single stranded 3′

region comprising a first primer region and wherein the complement of said first primer region is not present in said first adaptor, and the second adaptor has a single stranded 5′

region comprising a second primer region and wherein the complement of said second primer region is not present in said second adaptor, and wherein the first adaptor ligates to the fragments cut by the first restriction enzyme and the second adaptor ligates to the fragments cut by the second restriction enzyme; and

amplifying the adaptor-ligated fragments using a first primer complementary to said first primer region and a second primer comprising said second primer region to produce amplified fragments, wherein the adaptor-ligated fragments that contain both the first adaptor and the second adaptor are enriched in the amplified fragments relative to the adaptor-ligated fragments that contain the first adaptor and do not contain the second adaptor, and to the adaptor-ligated fragments that contain the second adaptor and do not contain the first adaptor;

providing a plurality of identical nucleic acid arrays consisting essentially of probes designed to detect the bases present at a plurality of polymorphisms predicted to be present in the second nucleic acid samples;

hybridizing each of the second nucleic acid samples to one of the plurality of identical nucleic acid arrays;

generating a plurality of hybridization patterns resulting from the hybridizations; and

analyzing the hybridization patterns to determine the bases present at the polymorphism in the population of individuals.

36. The method of claim 35 wherein the SNP is associated with the efficacy of a drug.