Capture reactions

• US 10,683,533 B2
• Filed: 01/24/2019
• Issued: 06/16/2020
• 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:
  US16/256,574
• Publication Number:
  US 20190233881A1
• Time in Patent Office: 509 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 improving performance of molecular inversion probe capture reactions, the method comprising:

obtaining a nucleic acid;

fragmenting the nucleic acid into a plurality of nucleic acid fragments to make a target available for capture;

introducing a plurality of molecular inversion probes (MIPs) to the plurality of nucleic acid fragments, wherein each of the plurality of MIPs hybridize to the target and captures a different one of a plurality of overlapping subregions of the target on the same strand;

circularizing the hybridized MIPs;

linearizing the circularized MIPs;

amplifying the circularized MIPs to generate amplicons; and

sequencing the amplicons.

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

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

4. The method of claim 1, wherein the plurality of 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. The method of claim 1, wherein the step of fragmenting comprises chemical fragmenting.

7. The method of claim 6, wherein chemical fragmenting comprises hydrolysis.

8. The method of claim 2, wherein the step of denaturing comprises heat-based denaturation.

9. The method of claim 1, wherein the amplification step further comprises introducing a differentiator tag sequence to the plurality of amplicons.

10. The method of claim 1, wherein each of the plurality of MIPs comprises at least one targeting arm, wherein the at least one targeting arm of each of the plurality of MIPs hybridizes to a different sequence of the target.