Enrichment through heteroduplexed molecules

• US 7,851,158 B2
• Filed: 12/28/2007
• Issued: 12/14/2010
• Est. Priority Date: 12/29/2006
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: McKernan, Kevin J.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US11/966,720
• Publication Number:
  US 20080274466A1
• Time in Patent Office: 1,082 Days
• Field of Search: 435/6, 435/91.2, 536/22.1, 536/23.1, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism

Abstract

The present invention relates to the enrichment of specific target sequences. Enrichment can be achieved through the formation of a heteroduplex that includes the specific target sequence and then the specific cleavage of the heteroduplex. A binding moiety is then added to the cleaved heteroduplex, allowing for the subsequent manipulation of the specific target sequence in the heteroduplex.

Claims

1. A method for the enrichment of a nucleic acid sequence, said method comprising:

forming a heteroduplex comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein said heteroduplex comprises at least one mismatched base pair between the first nucleic acid sequence and the second nucleic acid sequence;

circularizing the heteroduplex to form a circularized heteroduplex (CHET);

preferentially cutting the CHET to form a duplex using an enzyme that recognizes the mismatched base pair;

ligating a binding moiety nucleic acid sequence (BMNAS) into the duplex to form a binding moiety duplex, wherein said BMNAS comprises;

two nucleic acid sequences hybridized to one another and a binding moiety; and

selecting the binding moiety by using a purifying moiety to bind to the binding moiety, thereby enriching the second nucleic acid sequence.

2. The method of claim 1, wherein the preferential cutting occurs at a predetermined location relative to the mismatched base pair.

3. The method of claim 2, wherein Endo V is used to cut the CHET.

4. The method of claim 1, wherein the preferential cutting occurs at a location one base pair away from the mismatched base pair.

5. The method of claim 1, further comprising forming a nucleic acid homoduplex and circularizing the homoduplex to form a circularized homoduplex (CHOM), wherein the CHOM does not contain a mismatched base pair, and wherein said forming and circularizing of the homoduplex is performed together with the forming and circularizing of the heteroduplex.

6. The method of claim 5, wherein the step of preferentially cutting the CHET does not result in a significant cutting of the CHOM.

7. The method of claim 1, wherein the BMNAS further comprises a first restriction site.

8. The method of claim 7, wherein the BMNAS further comprises a second restriction site and wherein each of the restriction sites is located at opposite ends of the nucleic acid sequence.

9. The method of claim 1, wherein the binding moiety comprises biotin.

10. The method of claim 1, wherein more than one type of BMNAS is used, and wherein the BMNASs have degenerate 5′

and 3′

ends.

11. The method of claim 2, wherein at least 16 BMNASs are used, each BMNAS having a different 5′

and 3′

end sequence combination.

12. The method of claim 11, wherein at least 1024 BMNASs are used, each BMNAS having a different 5′

and 3′

end sequence combination.

13. The method of claim 1, further comprising the step of cutting the binding moiety duplex to form a cut binding moiety duplex (CBMD).

14. The method of claim 13, wherein the cutting is controlled by restriction sites within the BMNAS.

15. The method of claim 14, wherein the cutting occurs by a Type IIs or a Type III cutter.

16. The method of claim 15, wherein the cutting occurs approximately 30 base pairs away from a restriction site for the cutting.

17. The method of claim 13, further comprising the step of identifying the second nucleic acid sequence.

18. The method of claim 13, further comprising the step of identifying the mismatched base pair.

19. The method of claim 18, wherein said identification occurs by sequencing the mismatched base pair.

20. The method of claim 13, further comprising the step of adding a first adapter to a first end of the CBMD.

21. The method of claim 20, further comprising the step of adding a second adapter to a second end of the CBMD, wherein the first and second adapters are not the same.

22. The method of claim 21, wherein the first adapter is a first primer site and the second adapter is a second primer site.

23. The method of claim 22, further comprising the step of using the first and second primer sites to amplify a sequence between the two primer sites.

24. The method of claim 23, wherein the amplification is achieved through emulsion PCR.

25. The method of claim 23, further comprising the step of sequencing the amplified sequence.

26. The method of claim 1, wherein the method is performed in vitro.

27. The method of claim 1, wherein the enrichment of the second nucleic acid sequence occurs only through the binding of the binding moiety to the purifying moiety.

28. The method of claim 1, further comprising the step of forming a second CHET, wherein the first CHET and the second CHET have different mismatched base pairs.

29. The method of claim 28, wherein at least 100 CHETs are formed, each with a different mismatched base pair.

30. The method of claim 29, wherein at least 1000 CHETs are formed, each with a different mismatched base pair.

31. The method of claim 1, wherein the first nucleic acid comprises DNA.

32. The method of claim 1, wherein the second nucleic acid comprises DNA.

33. The method of claim 1, wherein there is more of the first nucleic acid sequence than the second nucleic acid sequence during the formation of the heteroduplex.

34. The method of claim 1, wherein the ratio of first to second nucleic acid sequences present during the formation of the heteroduplex is no less than 5:

1.

35. The method of claim 1, wherein the ratio of first to second nucleic acid sequences present during the formation of the heteroduplex is no less than 100:

1.

36. The method of claim 1, wherein the ratio of first to second nucleic acid sequences present during the formation of the heteroduplex is no less than 1000:

1.

37. A method for the enrichment of a nucleic acid sequence, said method comprising:

providing a sample containing a first nucleic acid sequence (FNAS) as well as a second nucleic acid sequence (SNAS);

providing two primers suitable for hybridization on complementary strands of the FNAS and the SNAS;

providing a polymerase;

combining the sample, the primers, and the polymerase to form a polymerase chain reaction mixture;

subjecting the polymerase chain reaction mixture to one or more polymerase chain reaction cycles to create a polymerase chain reaction extension product;

denaturing the polymerase chain reaction extension product to separate a FNAS and a SNAS;

annealing the polymerase chain reaction extension product to form a heteroduplex comprising the FNAS and the SNAS and to form a homoduplex comprising the FNAS;

circularizing the heteroduplex to form a circularized heteroduplex (CHET);

circularizing the homoduplex to form a circularized homoduplex (CHOM);

providing an endonuclease that preferentially cuts the heteroduplex at a location one base away from mismatched base pairs, wherein said cutting of the heteroduplex is preferential over the cutting of the homoduplex;

combining the CHET, the CHOM, and the endonuclease to form an endonuclease cleavage reaction mixture;

incubating the endonuclease cleavage reaction mixture so that the endonuclease preferentially cuts the CHET at a location one base away from a mismatched base pairs;

providing a plurality of binding moiety nucleic acid sequences (BMNAS), wherein said BMNASs comprise;

a double stranded nucleic acid sequence;

biotin associated with the double stranded nucleic acid sequence,

two EcoP15I restriction sites on each nucleic acid sequence, wherein one restriction site is located near or at the 3′

end of one nucleic acid strand and the second restriction site is located near or at the 5′

end of the same nucleic acid strand, and

degenerate ends on each end of each nucleic acid sequence;

ligating the BMNAS into the duplex to form a binding moiety duplex;

cutting the binding moiety duplex using EcoP15I;

concentrating the binding moiety duplex by using magnetic beads;

cutting the concentrated binding moiety duplex by using EcoP15I to form a cut binding moiety duplex (CBMD);

adding a first adapter to a first end of the CBMD;

adding a second adapter to a second end of the CBMD, wherein the first and second adapters are different;

performing emulsion PCR to amplify the CBMD using the first and second adapters as priming sites; and

performing highly parallel non-electrophoretic sequencing technique on the CBMD to identify the mismatched base pair.