Methods for analyzing nucleic acids

• US 9,677,124 B2
• Filed: 08/14/2015
• Issued: 06/13/2017
• Est. Priority Date: 03/14/2013
• Status: Active Grant

• Current Assignee: Molecular Loop Biosciences, Inc.
• Original Assignee: Good Start Genetics, Inc. (Invitae Corporation)
• Inventors: Umbarger, Mark
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US14/826,836
• Publication Number:
  US 20150354003A1
• Time in Patent Office: 669 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

The invention generally relates to methods for analyzing nucleic acids. In certain aspects, methods of the invention involve obtaining a sample including a nucleic acid template. A plurality of molecular inversion probes are tiled across a portion of the template. The probes are designed such that immediately adjacent probes hybridize to opposite strands of the nucleic acid template and probes on the same strand hybridize to the template in an overlapping manner. A region between targeting arms of a plurality of the molecular inversion probes is filled-in with nucleotides, and the filled-in region of a plurality of the probes is analyzed to obtain sequence information about the nucleic acid template.

Claims

1. A method for identifying an allelic status of a subject, the method comprising:

obtaining a sample comprising a nucleic acid template from a subject;

tiling a plurality of molecular inversion probes across both alleles of a locus on the template, wherein immediately adjacent probes hybridize to opposite strands of the nucleic acid template and probes on the same strand hybridize to the template in an overlapping manner;

filling in a region between targeting arms of a plurality of the molecular inversion probes with nucleotides;

analyzing the filled-in region of a plurality of the probes to obtain sequence information about both alleles of the locus; and

identifying an allelic status based on the sequence information.

2. The method of claim 1, wherein analyzing is by sequencing.

3. The method of claim 2, wherein prior to sequencing, the method further involves amplifying the filled-in region of the plurality of the probes.

4. The method of claim 2, wherein sequencing is sequencing-by-synthesis.

5. The method of claim 4, wherein sequencing-by-synthesis is single molecule sequencing-by-synthesis.

6. The method of claim 2, wherein sequencing information comprises a mutation in the nucleic acid sequence.

7. The method of claim 6, wherein the mutation is selected from the group consisting of:

a single nucleotide polymorphism (SNP);

an insertion;

a deletion;

an inversion;

a translocation;

a substitution; and

a combination thereof.

8. The method of claim 2, wherein prior to sequencing, a barcode is attached to the molecular inversion probe.

9. The method of claim 2, wherein prior to sequencing, a sequencing adaptor is attached to the molecular inversion probe.

10. The method of claim 2, wherein prior to sequencing, a barcode and a sequencing adaptor are attached to the molecular inversion probe.

11. The method of claim 2, wherein prior to sequencing, the molecular inversion probe is attached to a solid surface.

12. The method of claim 11, wherein the surface is a surface of a flow cell.

13. The method of claim 1, wherein the filling-in step uses a ligase.

14. The method of claim 1, wherein the filling-in step uses a polymerase.

15. The method of claim 13 or 14, wherein a ligase is used to covalently link the filled-in region to one of the targeting arms.