Asymmetric adapter library construction

• US 8,420,319 B2
• Filed: 09/11/2012
• Issued: 04/16/2013
• 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:
  US13/610,410
• Publication Number:
  US 20130029380A1
• Time in Patent Office: 217 Days
• Field of Search: 435/6.1, 435/91.1, 435/91.2, 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 asymmetrically tagged nucleic acid, comprising

(i) ligating an adaptor to both ends of a double-stranded nucleic acid fragment to produce a ligation product, wherein said adaptor is composed of a single strand of nucleic acid that comprises:

(a) a double stranded stem region and

(b) a loop region that comprises a cleavable linkage that is positioned between two non-complementary sequences; and

(ii) cleaving said ligation product using an agent that specifically cleaves said cleavable linkage, thereby producing an pair of asymmetrically tagged strands that each comprise a 5′

tag and a 3′

tag, where the sequences of the 5′

tag and the 3′

tag are different.

2. The method of claim 1, wherein said cleavable linkage is adjacent to at least one ribonucleotide.

3. The method of claim 2, wherein said cleaving is done using an RNAse.

4. The method of claim 1, wherein said RNAse is RNase 1.

5. The method of claim 1, wherein said cleavable linkage is a disulfide bond, and said cleaving is done using a reducing agent.

6. The method of claim 1, further comprising:

iii) copying an asymmetrically tagged strand of (ii) by;

(a) hybridizing a primer to the 3′

tag sequence of the asymmetrically tagged strand of (ii); and

(b) extending said primer using a polymerase.

7. The method of claim 6, wherein said polymerase is selected from the group consisting of:

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

8. The method of claim 1, wherein in said 5′

tag and/or 3′

tag 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.

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

10. 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 the ends of said adapter.

11. The method of claim 1, wherein said method further comprises isolating one strand of said pair of asymmetrically tagged strands.

12. The method of claim 11, wherein said isolating comprises treating said asymmetrically tagged nucleic acid strands with an exonuclease selected to digest only one strand of said asymmetrically tagged nucleic acid strands.

13. The method of claim 1, wherein said method further comprises:

digesting said pair of asymmetrically tagged nucleic acid strands with a restriction enzyme; 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.