METHODS AND SYSTEMS FOR UNIFORM ENRICHMENT OF GENOMIC REGIONS

• US 20090221438A1
• Filed: 02/23/2009
• Published: 09/03/2009
• Est. Priority Date: 04/24/2006
• Status: Active Grant

• Current Assignee: Roche NimbleGen, Inc. (Roche Holding AG)
• Original Assignee: Roche NimbleGen, Inc. (Roche Holding AG)
• Inventors: Kitzman, Jacob, Middle, Christina, D'Ascenzo, Mark, Albert, Thomas, Richmond, Todd, Jeddeloh, Jeffrey, Rodesch, Matthew
• Granted Patent: US 8,383,338 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase

Abstract

The present invention provides methods and compositions for the enrichment of target nucleic acids in a microarray system. In particular, the present invention provides methods and compositions for uniform enrichment of target nucleic acid molecules in a microarray format. The present invention also provides for intentionally non-uniform enrichment among target nucleic acid molecules.

Claims

1. A method for uniform enrichment of a population of nucleic acid molecules in a sample, comprising:

a) providing a sample of nucleic acid molecules comprising a plurality of target nucleic acid sequences,

b) hybridizing the sample to a support comprising immobilized nucleic acid probes under conditions to support hybridization between the immobilized nucleic acid probes and the plurality of target nucleic acid sequences, wherein said immobilized nucleic acid probes are complementary to said plurality of target nucleic acid sequences, and wherein the immobilized nucleic acid probes provide uniform hybridization among said plurality of target nucleic acid sequences, and

c) separating non-hybridized nucleic acid sequences from hybridized target nucleic acid sequences, thereby uniformly enriching a population of nucleic acid molecules in a sample.

2. The method of claim 1, wherein said separating comprises washing said support.

3. The method of claim 1, further comprising fragmenting said sample of nucleic acid molecules prior to said hybridizing.

4. The method of claim 3, further comprising ligating an adaptor molecule to one or both ends of a plurality of fragmented nucleic acid molecules prior to said hybridizing.

5. The method of claim 4, further comprising denaturing said sample of nucleic acid molecules prior to said hybridizing.

6. The method of claim 1, further comprising eluting a plurality of hybridized target nucleic acid sequences from the support.

7. The method of claim 6, further comprising sequencing the eluted target nucleic acid sequences.

8. The method of claim 1, wherein said support is a microarray slide.

9. The method of claim 1, wherein said support is a bead.

10. The method of claim 1, wherein said population of nucleic acid molecules is a population of genomic DNA molecules.

11. The method of claim 1, wherein said population of nucleic acid molecules is a population of amplified genomic DNA molecules.

12. The method of claim 1, wherein the probes are characterized in that the frequency of the individual sequences of said immobilized nucleic acid probes corresponds to the frequency of the corresponding plurality of target nucleic acid sequences within a population of nucleic acid molecules.

13. The method of claim 12, wherein determining the frequency comprises utilizing an empirically-fit linear regression model.

14. A composition comprising a solid support and a plurality of nucleic acid probes immobilized on said solid support, wherein each of said plurality of immobilized nucleic acid probes provides for uniform hybridization among a plurality of target nucleic acid sequences.

15. A kit for performing uniform enrichment of target nucleic acid sequences comprising one or more containers, wherein said one or more containers comprises:

a) a solid support comprising immobilized nucleic acid probes, wherein said probes are selected from a group consisting of a plurality of probes hybridizable to a plurality of target nucleic acid sequences and wherein said probes provide for uniform enrichment of said plurality of target nucleic acid sequences, and

b) one or more reagents for performing hybridizations, washes, and elution of target nucleic acid sequences.

16. A process for uniform enrichment of a population of nucleic acid sequences in a sample comprising a plurality of immobilized hybridization probe sequences wherein the frequency of the individual sequences of the immobilized hybridization probes corresponds to the frequency of a plurality of corresponding target nucleic acid sequences within a population of nucleic acid molecules, and wherein said process for uniform enrichment comprises hybridizing said probes to the corresponding target nucleic acid sequences and separating non-hybridized nucleic acid sequences from hybridized target nucleic acid sequences.

17. The process of claim 16, further comprising eluting said hybridized target nucleic acid sequences.

18. The process of claim 16, wherein the immobilized hybridization probes are immobilized on a microarray slide or a bead.

19. The process of claim 18, wherein determining the frequency comprises utilizing an empirically-fit linear regression model.

20. The process of claim 16, wherein said target nucleic acid sequences are genomic DNA sequences.