Method for constructing nucleic acid single-stranded cyclic library and reagents thereof

Patent metadata

Field	Value
Publication number	US-10351848-B2
Application number	US-201415510882-A
Country	US
Kind code	B2
Filing date	Nov 26, 2014
Priority date	Sep 12, 2014
Publication date	Jul 16, 2019
Grant date	Jul 16, 2019

Abstract

Provided are a method for constructing a nucleic acid single-stranded cyclic library and the reagents used therein. By the combination of interruption via a transposase with a restricted nick translation reaction, the method realizes a simple and rapid nucleic acid single-stranded cyclic library construction.

First claim

What is claimed is:

1. A method for constructing a library containing single-stranded cyclic nucleic acids, comprising:
randomly fragmenting a double-stranded nucleic acid sample with a transposase embedded complex, which comprises transposase and a first adaptor containing a transposase recognition sequence, to obtain fragmented double-stranded nucleic acids ligated with the first adaptor at each terminal thereof, with a gap between each 3′-end and the first adaptor;
ligating with a ligase a second adaptor to the fragmented double-stranded nucleic acid at the gap after removing the transposase from the reaction system, the second adaptor having a sequence different from that of the first adaptor;
performing a first PCR reaction with a first primer containing a uracil to obtain a first PCR product ligated with a first adaptor sequence and a second adaptor sequence respectively at two ends thereof;
enzyme-digesting the first PCR product with User enzyme at the uracil site to generate a gap, followed by double-stranded cyclization resulting in double-stranded cyclic nucleic acids;
subjecting the double-stranded cyclic nucleic acid, as a template, to constrained nick translation from the gap;
removing a portion without occurring the constrained nick translation in the double-stranded cyclic nucleic acids by digestion to obtain double-stranded linear nucleic acids;
ligating a third adaptor and an oligonucleotide adaptor sequence to the 3′-end and the 5′-end of each strand of the double-stranded linear nucleic acid, respectively;
performing a second PCR reaction with a second primer containing a first affinity marker at the 5′-end thereof, to obtain a second PCR product ligated with a third adaptor sequence and the oligonucleotide adaptor sequence respectively at two ends thereof;
capturing the second PCR product with a carrier having a second affinity marker capable of combining with the first affinity marker, and isolating single-stranded nucleic acids without the first affinity marker through nucleic acid denaturation; and
cyclizing the single-stranded nucleic acids without the first affinity marker with a single-stranded cyclization “bridge” sequence which is capable of combining with two ends of each single-stranded nucleic acid.

2. The method according to claim 1 , further comprising: digesting uncyclized single-stranded nucleic acids subsequent to cyclizing the single-stranded nucleic acids with the single-stranded cyclization “bridge” sequence.

3. The method according to claim 1 , wherein the transposase is removed from the reaction system by magnetic beads purification, column purification or chemical reagent treatment.

4. The method according to claim 1 , wherein a length of fragment generated during the constrained nick translation is controlled by an amount of dNTPs in the reaction system.

5. The method according to claim 1 , further comprising digesting uncyclized first PCR product prior to the constrained nick translation.

6. The method according to claim 1 , wherein removing a portion without occurring the constrained nick translation in the double-stranded cyclic nucleic acids by digestion further comprises:
digesting the double-stranded cyclic nucleic acids with a double-stranded exonuclease until a first gap between two ends of the internal strand encounters a second gap between two ends of the external strand within one double-stranded cyclic nucleic acid; followed by digesting a portion without occurring the constrained nick translation in each strand of thus obtained double-stranded cyclic nucleic acids with a single-stranded exonuclease.

7. The method according to claim 1 , further comprising:
end-repairing the double-stranded linear nucleic acid and dephosphorylating the 5′-end of each strand of the double-stranded linear nucleic acid prior to ligating the third adaptor to the 3′-end of each strand of the double-stranded linear nucleic acid.

8. The method according to claim 7 , comprising:
subjecting the dephosphorylated 5′-end of each strand of the double-stranded linear nucleic acid to phosphorylation subsequent to ligating the third adaptor to the 3′-end of each strand of the double-stranded linear nucleic acid; and
ligating the oligonucleotide adaptor sequence to the phosphorylated 5′-end of each strand of the double-stranded linear nucleic acid.

9. The method according to claim 1 , wherein the oligonucleotide adaptor sequence has a portion complementary with the third adaptor, wherein the portion is adjacent to the double-stranded linear nucleic acid.

10. The method according to claim 1 , wherein the first affinity marker is a biotin marker, and the second affinity marker is a streptavidin marker.

Assignees

• Mgi Tech Co Ltd

Inventors

• Geng Chunyu
• Chen Ruoying
• Jiang Yuan
• Zhao Xia
• Guo Rongrong
• He Lingyu
• Li Yaqiao
• Zhang Wenwei
• Jiang Hui
• Drmanac Radoje

Classifications

• C12N15/1093 Primary

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title

• C12N15/1089

  Design, preparation, screening or analysis of libraries using computer algorithms · CPC title

• C12Q1/6806

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

• C12Q1/6853

  using modified primers or templates · CPC title