METHODS AND SYSTEMS FOR SOLUTION BASED SEQUENCE ENRICHMENT

• US 20120046175A1
• Filed: 09/22/2011
• Published: 02/23/2012
• Est. Priority Date: 10/23/2007
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Inventors: Rodesch, Matthew, Albert, Thomas
• Granted Patent: US 9,790,543 B2
• Time in Patent Office: Days
• US Class Current: 506/1
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The present invention provides methods and systems for the capture and enrichment of target nucleic acids and analysis of the enriched target nucleic acids. In particular, the present invention provides for the enrichment of targeted sequences in a solution based format.

Claims

1. A method for isolating and reducing the complexity of a plurality of nucleic acid sequences, the method comprising, in order, the steps of:

a) providing;

i) a solid support wherein said solid support comprises hybridization probes immobilized on the support and hybridizable to target nucleic acid sequences, ii) a fragmented nucleic acid sample comprising target nucleic acid sequences,

b) amplifying said immobilized hybridization probes to produce amplification products that comprise a binding moiety, wherein said amplification products are maintained in solution,

c) hybridizing said nucleic acid sample to said amplification products in solution such that hybridization between said amplification products and target nucleic acid sequences is allowed to occur to produce hybridization complexes,

d) separating the target nucleic acid/amplification product hybridization complexes from non-specifically hybridized nucleic acids by said binding moiety, and

e) eluting the hybridized target nucleic acid sequences from the complexes thereby isolating and reducing the complexity of a plurality of nucleic acid sequences.

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

3. The method of claim 1, wherein said nucleic acid sample is a fragmented genomic DNA sample.

4. The method of claim 3, wherein said fragmented genomic DNA sample further comprises adaptor molecules at one or both ends of the fragments of genomic DNA sample.

5. The method of claim 1, wherein said hybridization probes further comprise primer binding sequences at one or both ends of said probes.

6. The method of claim 5, wherein said primer binding sequences when present at both ends of the probes are the same.

7. The method of claim 5, wherein said primer binding sequences when present at both ends of the probes are different.

8. The method of claim 1, wherein said amplifying comprises exponential polymerase chain reaction.

9. The method of claim 8, wherein said amplifying further comprises asymmetric polymerase chain reaction.

10. The method of claim 1, wherein said binding moiety is biotin.

11. The method of claim 10, wherein said separating comprises binding said biotin to a streptavidin coated substrate.

12. The method of claim 11, wherein said streptavidin coated substrate is a streptavidin coated paramagnetic particle.

13. The method of claim 1, further comprising washing said separated target nucleic acid/amplification product hybridization complexes prior to elution.

14. The method of claim 1, further comprising sequencing the eluted target nucleic acid sequences.

28. A kit comprising:

a) hybridization probe sequences comprising a binding moiety wherein said probe sequences are designed to hybridize to one or more target nucleic acid sequences and wherein said probe sequences are in solution, b) a substrate comprising a binding partner for binding said binding moiety, and c) instructions for performing the methods of claim 1.

29. The kit of claim 28, further comprising one or more of a hybridization solution, a wash solution and an elution solution.

30. The kit of claim 29, further comprising a magnet.

15. A method for isolating and reducing the complexity of a plurality of nucleic acid sequences the method comprising, in order, the steps of:

a) providing;

i) a solid support wherein said solid support comprises hybridization probes immobilized on the support and hybridizable to target nucleic acid sequences, ii) a nucleic acid sample comprising target nucleic acid sequences,

b) amplifying said immobilized hybridization probes to produce the amplification products that comprise a binding moiety, wherein said amplification products are maintained in solution,

c) hybridizing said nucleic acid sample to said amplification products in solution such that hybridization between said amplification products and target nucleic acids is allowed to occur to produce hybridization complexes,

d) separating the target nucleic acid/amplification product hybridization complexes from non-specifically hybridized nucleic acids by said binding moiety,

e) eluting the hybridized target nucleic acid sequences from the complexes thereby isolating and reducing the complexity of a plurality of nucleic acid sequences, and

f) sequencing the eluted target nucleic acid sequences.

16. The method of claim 15, wherein said solid support is a microarray slide.

17. The method of claim 15, wherein said nucleic acid sample is a fragmented genomic DNA sample.

18. The method of claim 17, wherein said fragmented genomic DNA sample further comprises adaptor molecules at one or both ends of the fragments of genomic DNA sample.

19. The method of claim 15, wherein said hybridization probes further comprise primer binding sequences at one or both ends of said probes.

20. The method of claim 19, wherein said primer binding sequences when present at both ends of the probes are the same.

21. The method of claim 19, wherein said primer binding sequences when present at both ends of the probes are different.

22. The method of claim 15, wherein said amplifying comprises exponential polymerase chain reaction.

23. The method of claim 22, wherein said amplifying further comprises asymmetric polymerase chain reaction.

24. The method of claim 15, wherein said binding moiety is biotin.

25. The method of claim 24, wherein said separating comprises binding said biotin to a streptavidin coated substrate.

26. The method of claim 25, wherein said streptavidin coated substrate is a streptavidin coated paramagnetic particle.

27. The method of claim 15, further comprises washing said separated target nucleic acid/amplification product hybridization complex prior to elution.