Methods for Genotyping with Selective Adaptor Ligation
First Claim
1. A method for reducing the complexity of a genomic DNA sample comprising:
- (a) fragmenting the sample with a restriction enzyme that has a recognition sequence that comprises at least one variable position, wherein the at least one variable position is in the single-stranded overhang generated by cleavage with the restriction enzyme and cleavage generates a plurality of overhangs of different sequence;
(b) ligating a selective adaptor to a subset of the fragments to generate adaptor-ligated fragments, wherein the 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); and
amplifying at least some of the adaptor-ligated fragments by polymerase chain reaction using a primer complementary to the selective adaptor.
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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.
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Citations
7 Claims
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1. A method for reducing the complexity of a genomic DNA sample comprising:
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(a) fragmenting the sample with a restriction enzyme that has a recognition sequence that comprises at least one variable position, wherein the at least one variable position is in the single-stranded overhang generated by cleavage with the restriction enzyme and cleavage generates a plurality of overhangs of different sequence; (b) ligating a selective adaptor to a subset of the fragments to generate adaptor-ligated fragments, wherein the 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); and amplifying at least some of the adaptor-ligated fragments by polymerase chain reaction using a primer complementary to the selective adaptor. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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Specification