Method of target enrichment and amplification
AssigneeAffymetrix, Inc.
InventorsXing Su, Shoulian Dong
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Abstract
The presently claimed invention provides methods and kits for amplifying a target sequence from within a nucleic acid population. The presently claimed invention provides selection probes which are complementary to at least a portion of said target sequence and mechanisms for adding a probe sequence to the 3′ end of a target sequence that is hybridized to a selection probe. The added 3′ probe sequence and a probe sequence added at the 5′ end of the target by adaptor ligation allow for selective amplification of the target sequence.
Claims17
A method of obtaining a population of target sequences, wherein each of said target sequences comprises a region of interest, comprising:
- fragmenting a sample population of nucleic acids to produce fragments;
- ligating an adaptor sequence to the 5′ ends of said fragments;
- combining said fragments with a collection of selection probes under conditions allowing for hybridization of complementary selection probes and target sequences to form probe-target complexes, each of said selection probes comprising a region that is complementary to a target sequence in said population of target sequences and further comprising a unique sequence at the 5′ end;
- extending the target sequences in the probe-target complexes using the selection probe in the probe-target complex as a template and thereby incorporating the complement of the unique sequence from the 5′ end of the selection probes into the 3′ end of the extended target sequences;
- and
- amplifying the extended target sequences using a first primer that is complementary to the extended region at the 3′ end of the extended target sequences and a second primer that is complementary to the adaptor region at the 5′ end of the extended target sequences.
A method of obtaining a population of target sequences, wherein each of said target sequences comprises a region of interest, comprising:
- fragmenting a reference nucleic acid sample to produce reference fragments;
- ligating a first and second adaptor to the ends of the reference fragments, at least one of said adaptors comprising both a promoter for in vitro transcription and a primer binding site, to generate a population of adaptor ligated reference fragments;
- making selection probes from said adaptor ligated reference fragments;
- fragmenting a sample population of nucleic acids to produce sample fragments;
- ligating a third adaptor sequence to the 5′ end of said sample fragments;
- combining said sample fragments with a collection of said selection probes under conditions allowing for hybridization of complementary selection probes and target sequences to form probe-target complexes, each of said selection probes comprising a region that is complementary to a target sequence in said population of target sequences and further comprising a unique sequence at the 5′ end;
- extending the target sequences in the probe-target complexes using the selection probe in the probe-target complex as a template and thereby incorporating the complement of the unique sequence from the 5′ end of the selection probes into the 3′ end of the extended target sequences;
- and
- amplifying the extended target sequences using a first primer to the extended region at the 3′ end of the extended target sequences and a second primer to the third adaptor region at the 5′ end of the extended target sequences.
Dependencies are inferred from the claim text.