Asymmetric adapter library construction

• US 8,883,990 B2
• Filed: 03/11/2013
• Issued: 11/11/2014
• Est. Priority Date: 04/30/2008
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Mikawa, Gi
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/794,170
• Publication Number:
  US 20140030704A1
• Time in Patent Office: 610 Days
• Field of Search: 536/23.1, 422/430
• US Class Current: 536/23.1
• 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 hairpin adaptor comprising:

a) a double stranded stem region; and

b) a loop region that comprises a cleavable linkage that is positioned between two non-complementary regions, wherein the non-complementary region that is 5′

of the cleavable linkage comprises a primer binding site that is in the range of 8 to 100 nucleotides in length.

2. The hairpin adaptor of claim 1, wherein primer binding site is in the range of 10-50 nucleotides in length.

3. The hairpin adaptor of claim 1, wherein the hairpin adaptor comprises one or more of the following:

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

4. The hairpin adaptor of claim 1, wherein the adaptor comprises a 5′

overhang.

5. The hairpin adaptor of claim 1, wherein the adaptor comprises a 3′

overhang.

6. The hairpin adaptor of claim 1, wherein the adaptor comprises a single nucleotide 3′

overhang.

7. The hairpin adaptor of claim 6, wherein the single nucleotide is a dT.

8. The hairpin adaptor of claim 1, wherein the cleavable linkage is enzymatically cleavable.

9. The hairpin adaptor of claim 8, wherein the cleavable linkage is cleavable by an RNase.

10. The hairpin adaptor of claim 9, wherein the RNAse is RNase 1.

11. The hairpin adaptor of claim 1, wherein the cleavable linkage is a disulfide bond, and the cleaving is done using a reducing agent.

12. A kit comprising:

the hairpin adaptor of claim 1; and

a primer that hybridizes to the primer binding site in the non-complementary region that is 5′

of the cleavable linkage.

13. The kit of claim 12, wherein the primer is in the range of 10-50 nucleotides in length.