Addition of an adaptor by invasive cleavage

• US 9,365,896 B2
• Filed: 08/09/2013
• Issued: 06/14/2016
• Est. Priority Date: 10/19/2012
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Ericsson, Olof John, Dahl, Fredrik, Johansson, Henrik, Srinivasan, Maithreyan
• Primary Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US13/963,723
• Publication Number:
  US 20140113296A1
• Time in Patent Office: 1,040 Days
• Field of Search: None
• US Class Current: 1/1
• 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 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/131 Inverse PCR

Abstract

This disclosure provides a method for adding an adaptor to a genomic sequence by invasive cleavage, as well as a kit for performing the method. In some embodiments, the method comprises: a) hybridizing genomic DNA to an adaptor comprising a double stranded region and a single stranded region comprising a 5′ overhang to produce a substrate for a flap endonuclease; b) cleaving the substrate using the flap endonuclease; c) ligating the recessed end of the double stranded region to the fragment to produce an adaptor-ligated DNA; d) intramolecularly ligating the ends of the adaptor-ligated DNA to produce a circular DNA molecule; and e) enzymatically processing the circular DNA molecule using an oligonucleotide that hybridizes to the adaptor and an enzyme. A kit for performing the method is also provided.

Claims

1. A method comprising:

a) hybridizing genomic DNA to an adaptor, wherein the adaptor comprises;

a double stranded region;

a single stranded region comprising a 5′

overhang; and

a nucleotide sequence for subsequent enzymatic processing;

wherein the 5′

overhang hybridizes to the genomic DNA to produce a substrate for a flap endonuclease, and wherein said 5′

overhang consists of a sequence of N nucleotides and/or nucleotide analogs that base pair with any nucleotide, where N is G, A, T or C;

b) cleaving said substrate using said flap endonuclease to produce a product comprising said adaptor and a flap endonuclease-generated fragment of said genomic DNA that is hybridized to said 5′

overhang and ligatable to the recessed end of said double stranded region, wherein said cleaving occurs at a random position in said genomic DNA;

c) ligating the recessed end of said double stranded region to said fragment to produce an adaptor-ligated DNA;

d) intramolecularly ligating the ends of said adaptor-ligated DNA to produce a circular DNA molecule; and

e) enzymatically processing said circular DNA molecule using an oligonucleotide that hybridizes to said adaptor and an enzyme.

2. The method of claim 1, wherein said enzymatically processing comprises primer extension, sequencing, amplification, and/or enzymatic cleavage.

3. The method of claim 1, wherein step e) comprises (i) amplifying said circular DNA molecule by PCR using inverse PCR primers that hybridize to said adaptor to produce an amplicon and (ii) sequencing said amplicon produced by (i).

4. The method of claim 1, wherein step e) comprises (i) amplifying said circular DNA molecule using rolling circle amplification to produce a product and (ii) sequencing said product of (i).

5. The method of claim 3, wherein said amplifying is done using one or more primers that bind to sites in said double stranded region.

6. The method of claim 3, wherein said amplifying is by clonal PCR.

7. The method of claim 3 wherein the products of step (i) are amplified by clonal PCR prior to sequencing.

8. The method of claim 1, wherein said double stranded region comprises a barcode sequence.

9. The method of claim 1, wherein said cleaving step b) and said ligating step c) are done in single reaction.

10. The method of claim 1, wherein said intramolecularly ligating is done using a single stranded ligase.

11. The method of claim 1, wherein said 5′

overhang consists of a sequence of N nucleotides.

12. The method of claim 1, wherein said nucleotide analogs are inosine.

13. The method of claim 1, wherein said 5′

overhang consists of a sequence of one or more N'"'"'s, and inosines, where the nucleotides adjacent to the double stranded region are N'"'"'s and the nucleotides distal to said double stranded region are inosines.

14. The method of claim 1, further comprising titrating the amount of said adaptor hybridized to said genomic DNA to produce fragments that have a desired average size.

15. The method of claim 1, wherein said genomic DNA is human genomic DNA.

16. The method of claim 1, wherein said double stranded region is 20-100 base pairs in length, and said single stranded region is 10-100 base pairs in length.

17. The method of claim 14, wherein the desired average size is between 100 bases and 10 kilobases (kb).

18. The method of claim 14, wherein the desired average size is between 200 bases and 1 kb.