Linker element and method of using same to construct sequencing library

Patent metadata

Field	Value
Publication number	US-10494630-B2
Application number	US-201415519112-A
Country	US
Kind code	B2
Filing date	Oct 14, 2014
Priority date	Oct 14, 2014
Publication date	Dec 3, 2019
Grant date	Dec 3, 2019

Abstract

Provided is a linker element and a method of using the linker element to construct a sequencing library, wherein the linker element consists of a linker A and a linker B, the linker A is obtained through the complementary pairing of a long nucleic acid strand and a short nucleic acid strand, the 5′ end of the long strand has a phosphoric acid modification, and the 3′ end of the short strand has an enclosed modification, with enzyme sites in the short strand; and the linker B is a nucleic acid single strand, and the 3′ end thereof can be in a complementary pairing with the 5′ end of the long strand of the linker A. Using the linker element of the present invention for constructing a sequencing library ensures the linking directionality of the linkers while solving the problems of fragment interlinking, linker self-linking and low linking efficiency, and reducing the purification reaction between steps, shortening the linking time and reducing costs.

First claim

The invention claimed is:

1. A method for constructing a sequencing library which uses a linker element consisting of a linker A and a linker B, wherein the linker A is generated from the complementary pairing of a long strand of nucleic acid and a short strand of nucleic acid, wherein the long strand has a phosphate modification at the 5′ end and the short strand has a blocking modification at the 3′ end, and has an enzyme active site in the short strand; and the linker B is a single-stranded nucleic acid, and the 3′ end thereof can be complementary to the 5′ end of the long strand of the linker A but the rest part cannot be complementary to the linker A,
wherein the method comprises the steps of:
(1) fragmenting a DNA to be tested;
(2) dephosphorylating and blunt-end repairing the DNA fragments obtained in step 1);
(3) linker ligations:
linker A ligation: the linker A is added to both ends of the DNA fragments obtained in Step (2) by a ligation reaction;
enzyme treatment and phosphorylation: depending on the enzyme active site in the short strand of the A linker, the DNA fragments ligated with the linker A are treated with the corresponding enzyme, and the unlinked 5′ ends of the fragments are phosphorylated; and
linker B ligation: through a ligation reaction, the linker B is added to both ends of the DNA fragments ligated with the linker A; and

(4) amplification of DNA fragments: polymerase chain reaction is carried out using the DNA fragments obtained in Step (3) as a template and using single-stranded nucleic acids C and D, which are complementary to the long strand of the linker A and the nucleic acid strand of the linker B, as primers,

upon which steps the sequencing library is obtained.

2. The method for constructing a sequencing library according to claim 1 , further comprising the steps of:
(5) hybridization capture: the product obtained in Step (4) is captured by hybridizing with an oligonucleotide probe and in the enrichment step of the hybridization product, a separation marker is introduced at the 5′ end of one strand of the double-stranded nucleic acid and a phosphate group modification is introduced at the 5′ end of the other strand; and
(6) separation and cyclization of single-stranded nucleic acids: the product obtained in Step (5) is separated by utilizing the separation marker to obtain another nucleic acid single strand without the separation marker; and a single strand circular nucleic acid product is obtained by cyclizing the obtained nucleic acid single strand, that is the sequencing library.

3. The method for constructing a sequencing library according to claim 1 , wherein in Step (2) the dephosphorylation is carried out by using shrimp alkaline phosphatase.

4. The method for constructing a sequencing library according to claim 1 , wherein in Step (5) the oligonucleotide probe is a library of oligonucleotide probes.

5. The method for constructing a sequencing library according to claim 1 , wherein in Step (2) the blunt-end repair is performed by using T4 DNA polymerase.

6. The method for constructing a sequencing library according to claim 1 , wherein in Step (5) the separation marker is a biotin modification.

7. The method for constructing a sequencing library according to claim 1 , wherein the long strand of linker A has a length of 40-48 bases and the short strand of linker A has a length of 9-14 bases.

Assignees

• Mgi Tech Co Ltd

Inventors

• Jiang Yuan
• Geng Chunyu
• Zhao Xia
• Fu Shujin
• He Lingyu
• Li Yaqiao
• Su Xiaoshan
• Wu Fanzi
• Zhang Wenwei
• Jiang Hui
• Alexeev Andrei
• Drmanac Radoje

Classifications

• C40B40/06

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title

• C12Q1/6855

  Ligating adaptors · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C40B50/06

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

• C12Q1/68

  involving nucleic acids · CPC title