Linker element and method of using same to construct sequencing library

• US 10,494,630 B2
• Filed: 10/14/2014
• Issued: 12/03/2019
• Est. Priority Date: 10/14/2014
• Status: Active Grant

• Current Assignee: MGI Tech Co., Ltd (BGI Genomics Co., Ltd.)
• Original Assignee: MGI Tech Co., Ltd (BGI Genomics 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
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US15/519,112
• Publication Number:
  US 20170233728A1
• Time in Patent Office: 1,876 Days
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/11 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 Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/162 Helper probe
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Y 207/01078 Polynucleotide 5'-hydroxyl-kinase (2.7.1.78)
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Y 301/03001 Alkaline phosphatase (3.1.3.1)
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/06 : Biochemical methods, e.g. using enzymes or whole viable microorganisms

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.

Claims

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.