Complexity management of genomic DNA

• US 7,993,883 B2
• Filed: 06/24/2010
• Issued: 08/09/2011
• Est. Priority Date: 10/27/1999
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Dong, Shoulian
• Primary Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US12/822,896
• Publication Number:
  US 20100292097A1
• Time in Patent Office: 411 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/91.2
• 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. A method of determining the bases present at nucleotide polymorphisms in a population of individuals comprising:

providing each of first nucleic acid samples from each of the individuals;

providing each of second nucleic acid samples comprising fragments, from said each of the first nucleic acid samples by;

fragmenting said each of the first nucleic acid samples using a first restriction enzyme and a second restriction enzyme, wherein the polymorphisms are predicted to be on fragments that are cut on their one end by the first restriction enzyme and on their other end by the second restriction enzyme; and

wherein the polymorphisms are single nucleotide polymorphisms (SNPs) and the fragments that are cut on their one end by the first restriction enzyme and on their other end by the second restriction enzyme are from said second nucleic acid samples;

producing adaptor-ligated fragments by ligating a first and a second adaptor to the fragments from said each of said second nucleic acid samples, wherein the first adaptor has a single stranded 3′

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

wherein adaptor-ligated fragments comprise overhangs having the first primer region and/or overhangs having the second primer region;

amplifying the adaptor-ligated fragments using a first primer complementary to said first primer region and a second primer comprising said second primer region and producing amplified fragments, wherein the amplified fragments generated from the adaptor-ligated fragments that contain both the first adaptor and the second adaptor are enriched in the amplified fragments relative to the amplified fragments generated from the adaptor-ligated fragments that contain the first adaptor and do not contain the second adaptor, and relative to the amplified fragments generated from 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 said amplified fragments to the plurality of identical nucleic acid arrays;

generating a plurality of hybridization patterns resulting from the hybridizing step; and

analyzing the hybridization patterns thereby determining the bases present at the polymorphisms in the population of individuals.

2. The method of claim 1 wherein the SNPs are associated with the efficacy of a drug.