Asymmetric adapter library construction

• US 8,029,993 B2
• Filed: 04/29/2009
• Issued: 10/04/2011
• 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:
  US12/432,080
• Publication Number:
  US 20090275087A1
• Time in Patent Office: 888 Days
• Field of Search: 435/6, 435/91.1, 536/23.1
• US Class Current: N/A
• 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 both ends of a double-stranded nucleic acid fragment, wherein said adapter comprises;

(a) a first and a second nucleic acid strand hybridized together, wherein said hybridized strands comprise one or more wobble base pair;

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

(d) a nucleic acid synthesis primer binding site positioned upstream of said one or more wobble base pair;

ii) annealing a synthesis primer specific for said nucleic acid synthesis primer binding site to said adaptor ligated fragment; and

iii) performing a first round of nucleic acid synthesis initiated from said annealed synthesis primer, wherein the nucleotide base incorporated at said one or more wobble base pair in the adapter region at a first end of said adaptor ligated fragment is different than the corresponding nucleotide in the adapter region at a second end of said adapter ligated fragment, thereby producing an asymmetrically tagged nucleic acid fragment.

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

3. The method of claim 2, wherein said one or more wobble base pair is positioned within said ligation site.

4. The method of claim 2, 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.

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

6. The method of claim 5, 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.

7. The method of claim 1, wherein said adapter further comprises one or more of the following:

a uniquely identifying tag (UID), an RNA polymerase promoter region, a primer binding site, a restriction enzyme site, and a recombination site.

8. An adapter comprising:

(a) a first and a second nucleic acid strand hybridized together, wherein said hybridized strands comprise one or more wobble base pair;

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

(d) a nucleic acid synthesis primer binding site positioned upstream of said one or more wobble base pair.

9. The adapter of claim 8, wherein said one or more wobble base pair is positioned within a restriction enzyme recognition and/or cut site in said adapter.

10. The method of claim 8, wherein said adapter further comprises one or more of the following:

a uniquely identifying tag (UID), an RNA polymerase promoter region, a primer binding site, a restriction enzyme site, and a recombination site.