SURFACE-CAPTURE OF TARGET NUCLEIC ACIDS

• US 20090156412A1
• Filed: 12/17/2007
• Published: 06/18/2009
• Est. Priority Date: 12/17/2007
• Status: Abandoned Application

• Current Assignee: Fluidigm Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Harris, Timothy D., Boyce, John J. IV
• Application Number:
  US11/958,173
• Publication Number:
  US 20090156412A1
• Time in Patent Office: Days
• US Class Current: 506/3
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The disclosure provides methods of capturing target nucleic acids (e.g., gene or gene fragments) onto a solid support for further analysis. The disclosed methods utilize a capture probe that selectively circularizes only the target nucleic acid. Following the circularization of the target, the linear, non-target, nucleic acids are removed from the sample. Next, the circularized target is linearized and bound to a solid support. To allow for linearization, the capture probe may include a cleavage site that can be a noncanonical nucleotide(s) (e.g., uracil in DNA) and/or a rare-cutter site (e.g., the Not I restriction site). In some embodiments, the target nucleic acid is captured onto a support without an intermediate amplification step.

Claims

1. A method of capturing a target nucleic acid onto a solid support, the method comprising:

(a) obtaining a sample comprising a target nucleic acid;

(b) circularizing the target nucleic acid;

(c) removing non-circularized nucleic acids;

(d) linearizing the target nucleic acid; and

(e) capturing the linearized target nucleic acid onto the solid support.

2. The method of claim 1, wherein the linearized target nucleic acid which is captured onto the solid support is unamplified.

3. The method of claim 1, wherein step (a) of obtaining the target nucleic acid comprises fragmenting a starting nucleic acid to produce the target nucleic acid having at least one defined end sequence.

4. The method of claim 3, wherein the average length of the target nucleic acid is at least 500 nts.

5. The method of claim 3, wherein the target nucleic acid contains (1) a unique combination of two defined ends or (2) a unique combination of one defined end sequence and one internal sequence.

6. The method of claim 1, wherein step (aa) comprises digesting the starting nucleic acid with one or more restriction enzymes.

7. The method of claim 1, wherein step (b) of circularizing the target nucleic acid comprises:

(ba) denaturing the target nucleic acid if it is double-stranded, thereby producing a single-stranded target nucleic acid.

(bb) contacting the single-stranded target nucleic acid with a double-stranded capture probe having two overhang ends specific to two corresponding sites on the target nucleic acid;

(bc) allowing the capture probe and the target nucleic acid to anneal to each other;

(bd) optionally, cleaving any branched structures; and

(be) ligating the capture probe and the target fragment to form a partially double-stranded closed circular nucleic acid.

8. The method of claim 7, wherein both overhang ends of the capture probe are complementary to two respective restriction cut sites of two different restriction enzymes.

9. The method of claim 1, wherein step (c) of removing the linear nucleic acids comprises treating the linear nucleic acids with an exonuclease.

10. The method of claim 1, wherein step (d) of linearizing the target nucleic acid comprises treating the circularized target nucleic acid with a rare-cutter restriction enzyme.

11. The method of claim 1, wherein step (d) of linearizing the target nucleic acid comprises treating the circularized target nucleic acid with glycosylase-lyase and endonuclease.

12. The method of claim 1, wherein step (d) of linearizing the target nucleic acid comprises treating the circularized target nucleic acid with uracil DNA glycosylase-lyase and endonuclease VIII.

13. The method of claim 1, wherein step (d) of linearizing the target nucleic acid comprises randomly fragmenting the linearized or circular single-stranded nucleic acid by shearing.

14. The method of claim 13, wherein the random fragments produced are of sufficient length to map back to a reference sequence.

15. The method of claim 1, wherein step (d) of linearizing the target nucleic acid is followed by adding a capture sequence to the linearized nucleic acid(s) at the 3′

end(s) if the capture sequence is absent.

16. The method of claim 15, wherein the capture sequence is polyN_n, wherein N is U, A, T, G, or C, and n≧

5.

17. The method of claim 1, wherein step (d) of linearizing the target nucleic acid is followed by adding a recognition site to the linearized nucleic acid(s) at the 5′

end(s) if the recognition site is absent.

18. The method of claim 1, wherein in step (e) the linearized nucleic acids are bound onto the solid support by hybridizing the capture sequence to a complementary sequence covalently attached to the solid support.

19. A method of sequencing a nucleic acid, comprising:

(i) capturing a target nucleic acid onto a solid support using the method of claim 1; and

(ii) sequencing the linearized nucleic acids captured on the solid support.

20. A method of determining a nucleic acid copy number, comprising:

(i) capturing an unamplified target nucleic acid onto a solid support using the method of claim 1; and

(ii) determining the copy number of the linearized nucleic acids captured on the solid support.

21. A method of capturing a nucleic acid onto a solid support, the method comprising:

(i) fragmenting a nucleic acid to produce one or more target fragments, each fragment having at least one defined end sequence;

(ii) denaturing the target fragment if it is double-stranded, thereby producing a single-stranded target fragment;

(iii) contacting the single-stranded target fragment with a double-stranded capture probe having two overhang ends specific to two corresponding sites on the target fragment;

(iv) allowing the capture probe and the target fragment to anneal to each other;

(v) optionally, cleaving any branched structures;

(vi) ligating the capture probe and the target fragment to form a closed circular nucleic acid;

(vii) removing remaining linear nucleic acids;

(viii) optionally, denaturing the double-stranded circular nucleic acid to create a single-stranded circular nucleic acid;

(ix) linearizing the single-stranded circular nucleic acid and, optionally, further fragmenting the linearized nucleic acid, or fragmenting the circular single-stranded nucleic acid;

(x) adding a capture sequence at the 3′

end(s) of the linearized nucleic acid fragment(s), and optionally adding a recognition site at the 5′

end(s) of the linearized nucleic acid fragment(s); and

(xi) capturing the linearized nucleic acids onto the solid support by hybridizing the capture sequence to a complementary sequence covalently attached to the solid support.

22. A nucleic acid probe comprising:

(a) a double-stranded nucleic acid having two overhang ends specific to two sites on a target nucleic acid, with one overhang end being complementary to a restriction cut site flanking a target sequence and the other end being complementary to a restriction cut site or an internal sequence;

(b) a cleavage site within the double-stranded nucleic acid of (a), said cleavage site selected from noncanonical nucleotide(s) and a rare-cutter site; and

(c) a capture sequence.

23. The probe of claim 22, wherein the capture sequence is polyN_n, wherein N is U, A, T, G, or C, and n≧

5.

24. The probe of claim 22, wherein the cleavage site comprises 1-10 uracils.

25. The probe of claim 22, wherein the probe comprises at least 1 uracil cleavage site in each strand of the double-stranded probe.