US 8,133,667 B2Granted

Methods for genotyping with selective adaptor ligation

AssigneeAffymetrix, Inc.

InventorsMichael H. Shapero, Keith W. Jones

Life of this patent

Estimating the term…

Loading patent…

Abstract

The present invention provides methods for reducing the complexity of a nucleic acid sample to interrogate a collection of target sequences. Complexity reduction can be accomplished by fragmenting the nucleic acid sample with a restriction enzyme that has at least one variable position in the recognition sequence. In some aspects adaptors that ligate to some but not all possible overhangs generated by digestion are ligated to the fragments. This selective adaptor ligation allows for selective amplification of a subset of the fragments using primers complementary to the adaptor sequence. In another aspect primers that are complementary to a subset of the fragments after adaptor ligation are used for amplification. Amplified fragments may be analyzed to genotype polymorphisms by hybridization to an array of probes that are complementary to target sequences that will be amplified.

Claims7

Dependent claims

A method for analyzing a genomic DNA sample comprising:

reducing the complexity of the sample by:
(a) fragmenting the sample with a restriction enzyme that has a recognition sequence that comprises at least one fixed position and has a variable region consisting of one or two variable positions, wherein the at least one fixed position and the variable region are in the single-stranded overhang generated by cleavage with the restriction enzyme and cleavage generates a plurality of single-stranded overhangs of different sequence including at least a first overhang and a second overhang;
(b) ligating a first selective adaptor comprising a single-stranded overhang that is perfectly complementary to the first overhang and a second selective adaptor comprising a single-stranded overhang that is perfectly complementary to the second overhang to a subset of the fragments to generate adaptor-ligated fragments, wherein the first selective adaptor is perfectly complementary to and ligates with a first subset of the single-stranded overhangs generated in step (a) and is not perfectly complementary to and does not ligate with a second subset of the single-stranded overhangs generated in step (a), wherein the second selective adaptor is perfectly complementary to and ligates with a third subset of the single-stranded overhangs generated in step (a) and is not perfectly complementary to and does not ligate with a fourth subset of the single-stranded overhangs generated in step (a) and wherein the first and second selective adaptors comprise a common priming sequence;
and
(c) amplifying at least some of the adaptor-ligated fragments by polymerase chain reaction using a primer that is complementary to the common priming sequence in the first and second selective adaptors to obtain amplified fragments that are a subset of the fragments;
and
analyzing the amplified fragments by hybridization to an array of probes.

4 more elements

Dependencies are inferred from the claim text.

Facts

Application: 12/272,370Filed 17 Nov 2008 · continuation
Priority: 29 Apr 2005
Published: US 2009/0075345 A119 Mar 2009
Granted: 13 Mar 2012
Term ends: Not estimatedThe source records don’t support an estimate.

Classification2 codes

C12Q 1/683
For detection of mutation or polymorphism: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms

Family

Application60/676,058Us provisional application · filed 29 Apr 2005
Application11/381,125Parent of this continuation · filed 1 May 2006
US 2009/0075345 A1Pre-grant publication · published 19 Mar 2009
US 7,452,671 B2Parent of this continuation
US 2006/0292597 A1Published application
US 8,133,667 B2This patent

From the US filing history. Foreign family members aren’t listed.