Methods of making di-tagged DNA libraries from DNA or RNA using double-tagged oligonucleotides

Patent metadata

Field	Value
Publication number	US-9243242-B2
Application number	US-201414226321-A
Country	US
Kind code	B2
Filing date	Mar 26, 2014
Priority date	May 8, 2011
Publication date	Jan 26, 2016
Grant date	Jan 26, 2016

Abstract

Disclosed are methods, compositions and kits related to making double-tagged DNA libraries from RNA/DNA samples. A double-tagged oligonucleotide (DTO) is employed to efficiently add two different tags to ends of DNAs to make a double-tagged DNA libraries. Also disclosed are methods to make mate pair libraries using the double-tagged oligonucleotide, and methods to make double-tagged single stranded DNA. The double-tagged DNA libraries of the invention are ready to be used on next generation sequencing machines.

First claim

What is claimed is:

1. A method of making a di-tagged single-stranded DNA from a double-stranded DNA, said method comprising the steps of:
a) providing a double-stranded double-tagged oligonucleotide (DTO) sequentially comprising a sequence tag A, a breaking site, a sequence tag B, and a single nick or gap in one of the two strands;
b) ligating two ends of said double-stranded DNA to said double-tagged oligonucleotide to generate a circular DNA-DTO;
c) removing the nicked/gapped strand using an exonuclease to generate a circular single-stranded DNA;
d) breaking said circular single-stranded DNA at said breaking site to generate a linear di-tagged single-stranded DNA.

2. The method of claim 1 , wherein said exonuclease comprises T7 exonuclease and exonuclease I.

3. The method of claim 1 , wherein said breaking site comprises a uracil nucleotide.

4. The method of claim 1 , further comprising performing a single primer PCR to amplify said di-tagged single-stranded DNA.

Assignees

• Wang Yan

Inventors

• Wang Yan

Classifications

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6855

  Ligating adaptors · CPC title

• C12N15/66

  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 title