Asymmetric adapter library construction

• US 8,288,097 B2
• Filed: 09/01/2011
• Issued: 10/16/2012
• Est. Priority Date: 04/30/2008
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Mikawa, Gi
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/224,134
• Publication Number:
  US 20120083017A1
• Time in Patent Office: 411 Days
• Field of Search: 435/6.1, 435/91.1, 536/23.1
• US Class Current: 435/6.1
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

The present invention provides methods and compositions for asymmetrically tagging a nucleic acid fragment using asymmetric adapters.

Claims

1. A method of producing an asymmetrically tagged nucleic acid fragment, said method comprising:

i) ligating an adapter to each end of a double-stranded nucleic acid fragment, wherein said adapter comprises;

(a) a first and a second nucleic acid strand associated with each other via one or more complementary domains, said adapter having a first end and a second end;

(b) one or more region of substantial non-complementarity between said first and second nucleic acid strands;

(c) a ligation site positioned on said first end of said adapter configured to allow ligation of said adapter to said double stranded nucleic acid fragment; and

(d) a hairpin structure positioned on the 3′

strand on said second end of said adaptor, said hairpin structure producing a nucleic acid synthesis self-priming site; and

ii) performing a first round of nucleic acid synthesis initiated from said self-priming site using a nucleotide polymerase, thereby producing an asymmetrically tagged nucleic acid fragment.

2. The method of claim 1, wherein said nucleotide polymerase is selected from the group consisting of:

a RNA polymerase, a mesophilic DNA polymerase, a reverse transcriptase, and a thermophilic DNA polymerase.

3. The method of claim 1, wherein in said region of substantial non-complementarity, said first and/or second nucleic acid strands comprise one or more of the following:

a unique identifier (UID), an RNA polymerase promoter region, a primer binding site, a restriction enzyme site, and a recombination site.

4. The method of claim 1, wherein said adaptor comprises an RNA polymerase promoter region adjacent to said hairpin structure or within the duplex region of said hairpin structure.

5. The method of claim 4, wherein said RNA promoter region is oriented such that RNA polymerization proceeds toward said hairpin structure.

6. The method of claim 1, wherein said double-stranded nucleic acid fragment is produced by digesting a parent double-stranded nucleic acid sample with a restriction enzyme and polishing the ends of the resultant restriction enzyme fragments to create ends compatible with said ligation site of said adapter.

7. The method of claim 1, wherein said method further comprises isolating one strand of said asymmetrically tagged nucleic acid fragment.

8. The method of claim 7, wherein said isolating comprises treating said asymmetrically tagged nucleic acid fragment with an exonuclease selected to digest only one strand of said asymmetrically tagged nucleic acid fragment.

12. The method of claim 1, wherein said adapter comprises one or more wobble base pair positioned in said one or more complementary domains such that said resultant asymmetrically tagged nucleic acid fragment comprises a restriction enzyme recognition and/or cut site at only one end.

13. The method of claim 12, wherein said one or more wobble base pair is positioned within said ligation site.

14. The method of claim 12, wherein said method further comprises:

digesting said asymmetrically tagged nucleic acid fragment with a restriction enzyme specific for said restriction enzyme recognition and/or cut site; and

ligating a second, different adapter to said digested fragment, said second adapter having a ligation site compatible with the digested end of said fragment.

16. The method of claim 1, wherein said adapter further comprises a cleavable linker in the loop region of said hairpin structure.

9. A nucleic acid adapter comprising:

(a) a first and a second nucleic acid strand associated with each other via one or more complementary domains, said adapter having a first end and a second end;

(b) one or more region of substantial non-complementarity between said first and second nucleic acid strands;

(c) a ligation site positioned on said first end of said adapter configured to allow ligation of said adapter to a double stranded nucleic acid fragment; and

(d) a hairpin structure positioned on the 3′

strand on said second end of said adaptor, said hairpin structure producing a nucleic acid synthesis self-priming site.

10. The adapter of claim 9, wherein said region of substantial non-complementarity in said first or second nucleic acid strand comprises one or more of the following:

a unique identifier (UID), an RNA polymerase promoter region, a primer binding site, a restriction enzyme site, and a recombination site.

11. The adapter of claim 9, wherein said adaptor comprises an RNA polymerase promoter region adjacent to said hairpin structure or within the duplex region of said hairpin structure.

15. The adapter of claim 9, wherein said adapter further comprises one or more wobble base pair positioned in said one or more complementary domains within a restriction enzyme recognition and/or cut site in said adapter.

17. The adapter of claim 9, wherein said adapter further comprises a cleavable linker in the loop region of said hairpin structure.