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

Patent metadata

Field	Value
Publication number	US-10023906-B2
Application number	US-201415510904-A
Country	US
Kind code	B2
Filing date	Oct 14, 2014
Priority date	Sep 12, 2014
Publication date	Jul 17, 2018
Grant date	Jul 17, 2018

Abstract

Provided in the present invention are a method for constructing a nucleic acid single-stranded cyclic library and reagent kit thereof. The method comprises the steps of using a transposase embedding complex to randomly break nucleic acids and connect a first linker; connecting a second linker at a gap; performing a first PCR reaction, wherein the 5′ end of one of the primers has a first affinity tag, resulting in a product with two ends connected to different linker sequences; binding the product to a solid vector having a second affinity tag; degenerating and separating single strands having no affinity tag; and cyclizing the single strands.

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 transposases 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, resulting in a gap between each 3′-end and the first adaptor;
ligating with 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 pair of primers targeting the first adaptor and the second adaptor respectively, to obtain a first PCR product ligated with a first adaptor sequence and a second adaptor sequence respectively at two ends thereof, wherein one of the first pair of primers contains a first affinity marker at the 5′-end thereof;
contacting the first PCR product with a solid carrier having a second affinity marker, such that the first affinity marker is combined with the second affinity marker;
isolating single-stranded nucleic acids without the first affinity marker through denaturing the first PCR product combined with the solid carrier; and
cyclizing the single-stranded nucleic acid with a single-stranded cyclization “bridge” sequence which is capable of combining with two ends of the single-stranded nucleic acid.

2. The method according to claim 1 , further comprising: performing, prior to the first PCR reaction, a second PCR reaction with a second pair of primers targeting the first adaptor and the second adaptor respectively, to obtain a second PCR product ligated with the first adaptor sequence and the second adaptor sequence respectively at two ends thereof.

3. The method according to claim 2 , wherein one of the second pair of primers contains a sample-tag sequence at the 5′-end thereof.

4. The method according to claim 2 , further comprising a step subsequent to the second PCR reaction and prior to the first PCR reaction: capturing a single-stranded nucleic acid containing an exon sequence, used for the first PCR reaction, from the second PCR product with a probe for the exon sequence, wherein the probe for the exon sequence contains the first affinity marker and is capable of combining with the second affinity marker of the solid carrier.

5. The method according to claim 4 , further comprising: blocking a primer sequence located at each end of single strand of the second PCR product with a primer blocking sequence, prior to capturing the single-stranded nucleic acid containing the exon sequence of the second PCR product with the probe for the exon sequence.

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

7. 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.

8. The method according to claim 1 , wherein the solid carrier is magnetic beads.

9. The method according to claim 1 , 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, resulting in 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 second PCR reaction with a second pair of primers targeting the first adaptor and the second adaptor respectively, to obtain a second PCR product ligated with a first adaptor sequence and a second adaptor sequence respectively at two ends thereof, wherein one of the second pair of primers contains a sample-tag sequence at the 5′-end thereof;
blocking a primer sequence located at each end of single-strand of the second PCR product with a primer blocking sequence;
capturing a single-stranded nucleic acid containing an exon sequence from the second PCR product with a probe for the exon sequence containing a biotin marker and capable of combining with a streptavidin marker of the solid carrier;
performing a first PCR reaction with a first pair of primers targeting two ends of the single-stranded nucleic acid containing the exon sequence respectively, to obtain a first PCR product ligated with different adaptor sequences respectively at two ends thereof, wherein one of the first pair of primers contains the biotin marker at the 5′-end thereof;
contacting the first PCR product with the solid carrier having a streptavidin marker, such that the biotin marker is combined with the streptavidin marker;
isolating single-stranded nucleic acids without the biotin marker through denaturing the first PCR product combined with the solid carrier;
cyclizing the single-stranded nucleic acid without the biotin marker with a single-stranded cyclization “bridge” sequence which is capable of combining with the two ends of the single-stranded nucleic acid without the biotin marker.

10. The method according to claim 3 , further comprising a step subsequent to the second PCR reaction and prior to the first PCR reaction: capturing a single-stranded nucleic acid containing an exon sequence, used for the first PCR reaction, from the second PCR product with a probe for the exon sequence, wherein the probe for the exon sequence contains the first affinity marker and is capable of combining with the second affinity marker of the solid carrier.

Assignees

• Bgi Shenzhen Co Ltd
• Mgi Tech Co Ltd

Inventors

• Geng Chunyu
• Guo Rongrong
• Chen Ruoying
• He Lingyu
• Zhang Wenwei
• Jiang Hui

Classifications

• C12Q2525/191

  incorporating an adaptor · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C40B40/08

  Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries · CPC title

• C40B60/14

  for creating libraries · CPC title

• C40B50/06

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title