CAPTURE REACTIONS

• US 20130274146A1
• Filed: 04/17/2012
• Published: 10/17/2013
• Est. Priority Date: 04/16/2012
• Status: Active Grant

• Current Assignee: Molecular Loop Biosciences, Inc.
• Original Assignee: Good Start Genetics, Inc. (Invitae Corporation)
• Inventors: Umbarger, Mark, Porreca, Gregory, Church, George, Towne, Charles
• Granted Patent: US 10,227,635 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• 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 sample;

fragmenting the nucleic acid into nucleic acid fragments;

introducing a plurality of molecular inversion probes into the sample, wherein each probe is specific for a different target and each probe comprises a first targeting arm and a second targeting arm joined to a universal central region;

capturing multiple targets within the sample with two or more of the molecular inversion probes, the multiple targets being on one or more of the nucleic acid fragments; and

amplifying two or more of the targets in a single reaction vessel.

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

7. The method of claim 6, wherein the chemical fragmenting comprises pH-based shearing.

8. The method of claim 7, wherein the pH-based shearing also denatures the nucleic acid.

9. The method of claim 2, wherein the step of denaturing comprises pH-based denaturing.