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US 9,790,543 B2Granted
Methods and systems for solution based sequence enrichment
AssigneeRoche Sequencing Solutions, Inc.
InventorsMatthew Rodesch, Thomas Albert
Life of this patent
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Abstract
The present invention provides methods and systems for the capture and enrichment of target nucleic acids and analysis of the enriched target nucleic acids. In particular, the present invention provides for the enrichment of targeted sequences in a solution based format.
Claims29
A method for reducing the genetic complexity of a plurality of nucleic acid molecules, the method comprising, in order, the steps of:
- a) providing a plurality of support-immobilized oligonucleotide probes defining a complete sequence of at least one genetic locus having a size of at least 100 kb;
- b) amplifying said support-immobilized oligonucleotide probes in solution in the presence of a nucleotide containing a binding moiety to generate a pool of amplification products derived from the support-immobilized oligonucleotide probes, wherein the pool of amplification products contain the binding moiety, and wherein the pool of amplification products are maintained in solution;
- c) contacting under aqueous conditions the pool of amplification products generated in step b) to a sample that comprises a plurality of fragmented nucleic acid molecules, a subset of the plurality of fragmented nucleic acid molecules being target nucleic acid molecules having target nucleic acid sequences;
- d) capturing the target nucleic acid molecules in hybridization complexes with the pool of amplification products, by providing a binding partner for the binding moiety;
- e) separating the hybridization complexes from unbound and non-specifically bound nucleic acids;
- and
- f) eluting the captured target nucleic acid molecules from the hybridization complexes in an eluate pool having reduced genetic complexity relative to the sample, wherein the eluate pool is enriched for the target nucleic acid molecules.
4 more elements
A method for reducing the genetic complexity of a plurality of nucleic acid molecules, the method comprising, in order, the steps of:
- a) amplifying into solution, in the presence of a nucleotide covalently linked to a binding moiety, a plurality of support-immobilized oligonucleotide probes hybridizable to target nucleic acid sequences, to produce, in solution, amplification products comprising the binding moiety;
- b) purifying the amplification products away from the support-immobilized oligonucleotide probes;
- c) contacting the amplification products, under aqueous hybridizing conditions, to a sample that comprises the plurality of fragmented nucleic acid molecules, a subset of the plurality being target nucleic acid molecules having the target nucleic acid sequences, to capture the target nucleic acid molecules in hybridization complexes with the amplification products;
- d) providing a binding partner for the binding moiety and separating the hybridization complexes from unbound and non-specifically bound nucleic acids;
- and
- e) eluting the captured target nucleic acid molecules from the hybridization complexes in an eluate pool comprising the plurality of nucleic acid molecules having reduced genetic complexity relative to the sample, wherein the eluate pool is enriched for target nucleic acid molecules as compared to a sample not having reduced genetic complexity.
3 more elements
Dependencies are inferred from the claim text.