Methods for genotyping selected polymorphism

• US 9,388,461 B2
• Filed: 06/13/2014
• Issued: 07/12/2016
• Est. Priority Date: 08/05/2003
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Jones, Keith W., Shapero, Michael H.
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US14/303,954
• Publication Number:
  US 20150010905A1
• Time in Patent Office: 760 Days
• Field of Search: 435/91.2, 435/91.21
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Methods for genotyping polymorphisms using a locus specific primer that is complementary to a region near a selected polymorphism are described. Methods for synthesizing pools of locus specific primers that incorporate some degenerate positions are also disclosed. A plurality of different sequence capture probes are synthesized simultaneously using degenerate oligonucleotide synthesis. The sequence of the locus specific regions of the capture probes are related in that they have some bases that are identical in each sequence in the plurality of sequences and positions that vary from one locus specific region to another. The sequences are selected based on proximity to a polymorphism of interest and because they conform to a similar sequence pattern.

Claims

1. A method for amplifying a collection of target sequences from a nucleic acid sample wherein each target sequence comprises a common sequence and a consensus sequence immediately adjacent to the common sequence, the method comprising:

fragmenting the nucleic acid sample into fragments with a selected restriction enzyme;

ligating an adaptor to the fragments to obtain adaptor-ligated fragments, wherein said adaptor comprises a first priming sequence;

contacting the adaptor-ligated fragments with probes, wherein each probe comprises a second priming sequence and a target specific sequence that comprises a region that is complementary to the common sequence and a region that is complementary to the consensus sequence;

extending at least some of the probes; and

amplifying the extended probes with primers to said first and second priming sequences.

2. The method of claim 1, wherein labeled nucleotides are incorporated into the extended probes and extended probes are isolated by affinity chromatography.

3. The method of claim 2, wherein said labeled nucleotides are labeled with biotin and, wherein avidin, streptavidin or an anti-biotin antibody is used to isolate extended probes.

4. The method of claim 1, wherein prior to amplification the extended probes are made double stranded and single stranded nucleic acid in the sample is digested with a nuclease.

5. The method of claim 1, wherein there are 1,000 to 5,000 different target sequences in the collection of target sequences.

6. The method of claim 1, wherein there are 2,000 to 10,000 different target sequences in the collection of target sequences.

7. The method of claim 1, wherein there are 10,000 to 1,000,000 different target sequences in the collection of target sequences.