TARGET ENRICHMENT AND LABELING FOR MULTI-KILOBASE DNA

• US 20130323725A1
• Filed: 03/14/2013
• Published: 12/05/2013
• Est. Priority Date: 06/01/2012
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: ACH, ROBERT A., PETER, BRIAN JON, TIMAR, ZOLTAN, MYERSON, JOEL, SAMPSON, JEFFREY ROBERT, HOGREFE, HOLLY
• Granted Patent: US 10,308,979 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• 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 2521/501 Ligase
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

This disclosure provides a method comprising: a) clamping the top and bottom strands of a double stranded DNA molecule to produce a duplex in which the top and bottom strands are linked; b) denaturing the duplex to produce a denatured product; and c) renaturing the denatured product in the presence of a labeled oligonucleotide that is complementary to a sequence of nucleotides in the double stranded DNA molecule, thereby producing a D-loop-containing product. Kits for performing the method and products made by the method are also provided.

Claims

1. A method comprising:

a) clamping the top and bottom strands of a double stranded DNA molecule to produce a duplex in which the top and bottom strands are linked;

b) denaturing the duplex to produce a denatured product; and

c) renaturing the denatured product in the presence of a labeled oligonucleotide that is complementary to a sequence of nucleotides in the double stranded DNA molecule, thereby producing a D-loop-containing product.

2. The method of claim 1, wherein the clamping comprises covalently linking the top and bottom strands of the double stranded DNA molecule to produce a duplex in which the top and bottom strands are covalently linked.

3. The method of claim 2, wherein the covalently linking is done by ligating a hairpin adaptor to one or both ends of the double stranded DNA.

4. The method of claim 3, wherein the hairpin adaptor comprises a hairpin region that has a T_m of over 70°

C.

5. The method of claim 2, wherein the covalently linking is done by ligating a double stranded adaptor to one or both ends of the double stranded DNA, and covalently cross-linking the top and bottom strands of the double stranded adaptor.

6. The method of claim 2, wherein the covalently linking is done by covalently linking the top and bottom strands of the double stranded DNA molecule to one another directly.

7. The method of claim 2, wherein the covalently linking is done by adding a terminal nucleotide that comprises a linking moiety to the top or bottom strand of the double stranded DNA molecule, and then reacting the linking moiety with the other strand of the double stranded DNA molecule.

8. The method of claim 1, wherein the clamping comprises non-covalently linking the top and bottom strands of the double stranded DNA molecule using a protein that holds the top and bottom strands together.

9. The method of claim 1, wherein the labeled oligonucleotide comprises an affinity tag.

10. The method of claim 9, wherein the capture moiety is a biotin moiety.

11. The method of claim 1, wherein the labeled oligonucleotide comprises an optically detectable label.

12. The method of claim 11, wherein the optically detectable label is a fluorescent label.

13. The method of claim 1, wherein the labeled oligonucleotide is made of LNA or UNA.

14. The method of claim 9, further comprising:

d) contacting the D-loop-containing product with a solid support comprising a surface tethered capture agent for the affinity tag, thereby binding the D-loop-containing product to the solid support and isolating the D-loop-containing product from other nucleic acids in the sample.

15. The method of claim 14, wherein the method comprises genotyping the double stranded DNA molecule in the isolated D-loop-containing product.

16. The method of claim 14, wherein one or more labeled oligonucleotides hybridize to more than one region of the double stranded DNA molecule, and the method comprises supercoiling the D-loop-containing product after it is contacted with the solid support.

17. The method of claim 14, wherein the covalently linking is done by ligating an adaptor to the double stranded DNA, wherein the adaptor comprises at least one cleavable linkage so that it can be removed from the D-loop-containing product after the D-loop-containing product has been isolated.

18. The method of claim 2, wherein the covalently linking is done by ligating an adaptor to the double stranded DNA, and wherein the adaptor comprises a nucleotide sequence that produces a restriction site if primer dimers are produced.

19. A composition of matter comprising:

a D-loop containing product comprising a double stranded DNA molecule in which the top and bottom strands are clamped together, and a labeled oligonucleotide annealed a sequence in said double stranded DNA molecule.

20. A kit comprising:

reagents for clamping the top and bottom strands of a double stranded DNA molecule; and

a labeled oligonucleotide, wherein the labeled oligonucleotide is complementary to a sequence of nucleotides in the double stranded DNA molecule.