Capture reactions

• US 10,227,635 B2
• Filed: 04/17/2012
• Issued: 03/12/2019
• Est. Priority Date: 04/16/2012
• Status: Active Grant

• Current Assignee: Molecular Loop Biosciences, Inc.
• Original Assignee: Molecular Loop Biosolutions, LLC
• Inventors: Umbarger, Mark, Porreca, Gregory, Towne, Charles, Church, George
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US13/448,961
• Publication Number:
  US 20130274146A1
• Time in Patent Office: 2,520 Days
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions

Abstract

The invention generally relates to methods of performing a capture reaction. In certain embodiments, the method involves obtaining a nucleic acid, fragmenting the nucleic acid, and capturing a target sequence on the nucleic acid fragment using a capture moiety, such as a molecular inversion probe.

Claims

1. A method of performing a molecular inversion probe capture reaction, the method comprising:

denaturing a nucleic acid;

fragmenting the nucleic acid into nucleic acid fragments in order to expose a target site for a molecular inversion probe that was not exposed prior to fragmenting, wherein the fragmenting step occurs during the denaturing step, and wherein the nucleic acid is fragmented and denatured by heating the nucleic acid in a buffer system to shift a pH of the buffer system;

introducing a plurality of molecular inversion probes under conditions such that the molecular inversion probe hybridizes to the target site on the nucleic acid fragment, and wherein each of the plurality of molecular inversion probes is configured to capture a different subregion of the target site, and wherein each subregion is different and overlaps with at least one other subregion on the same strand;

circularizing the hybridized molecular inversion probe to form a circularized probe/target nucleic acid molecule;

isolating the circularized probe/target nucleic acid molecule;

linearizing the circularized probe/target nucleic acid molecule, thereby forming a linearized probe/target nucleic acid molecule;

conducting an amplification reaction with the linearized probe/target nucleic acid molecule to generate an amplification product, wherein the amplification reaction introduces a barcode sequence to the amplification product; and

sequencing the amplification product.

2. The method of claim 1, further comprising the step of denaturing the nucleic acid or at least one nucleic acid fragment prior to the capturing step.

3. The method of claim 1, wherein the nucleic acid fragments are from about 5 kb to about 100 kb in length.

4. The method of claim 1, wherein the nucleic acid fragments are from about 1 kb to about 10 kb in length.

5. The method of claim 1, wherein the nucleic acid is selected from the group of genomic DNA, genomic RNA, whole or partial genome amplification product, high molecular weight DNA, and high molecular weight RNA.

6. A method of improving performance of capture reactions using molecular inversion probes having two targeting arms, the method comprising:

denaturing a nucleic acid;

fragmenting the nucleic acid into nucleic acid fragments in order to expose a target site having one or more bases to molecular inversion probes that were not exposed prior to fragmenting, the molecular inversion probes being designed such that each molecular inversion probes is configured to capture a different subregion of the target site, and wherein each subregion is different and overlaps with at least one other subregion on the same strand, wherein the fragmenting step occurs during the denaturing step, and wherein the nucleic acid is fragmented and denatured by heating the nucleic acid in a buffer system to shift a pH of the buffer system;

introducing the molecular inversion probes under conditions such that the molecular inversion probes hybridize to the target site on the nucleic acid fragment;

circularizing the hybridized molecular inversion probes to form circularized probe/target nucleic acid molecules;

isolating the circularized probe/target nucleic acid molecules;

linearizing the circularized probe/target nucleic acid molecules, thereby forming linearized probe/target nucleic acid molecules;

conducting an amplification reaction with the linearized probe/target nucleic acid molecules to generate one or more amplification products, wherein the amplification reaction introduces a barcode sequence to the amplification products; and

sequencing the amplification products; and

wherein at least one targeting arm of each of the more than one molecular inversion probe does not hybridize to a same sequence of the nucleic acid.