METHODS AND KITS OF CONSTRUCTING SEQUENCE LIBRARY FOR USE IN DETECTING CHROMOSOME COPY NUMBER VARIATION

• US 20200131507A1
• Filed: 12/11/2017
• Published: 04/30/2020
• Est. Priority Date: 06/21/2017
• Status: Abandoned Application

• Current Assignee: Berry Genomics Co., Ltd.
• Original Assignee: Berry Genomics Co., Ltd.
• Inventors: Shang, Ling, Lv, Meng, Chen, Xiangbin, Wang, Tingting, Zhang, Jianguang
• Application Number:
  US16/624,873
• Publication Number:
  US 20200131507A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2547/101 by confinement to a single tube/container
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

Provided herein is a method of constructing a high throughput sequencing library for use in detecting chromosome copy number variation comprising mainly the following steps: (1) subjecting DNAs to be tested to random fragmentation by a double-strand DNA fragmentation enzyme; (2) end-filling and adding poly-adenine at the 3′end of the fragmented DNAs; (3) connecting the end-filled DNAs having a 3′end poly-adenine with sequencing linkers to obtain connected products; (4) purifying the connected products to obtain the sequencing library; wherein steps (1)-(3) are performed in a single reaction tube. Also provided is a kit for constructing a sequencing library for use in detecting chromosome copy number variation.

Claims

1. A method of rapidly constructing a high throughput sequencing library for use in detecting chromosome copy number variation, characterized in that, the method comprises the following steps:

(1) subjecting DNAs to be tested to random fragmentation by a double-strand DNA fragmentation enzyme;

(2) end-filling and adding poly-adenine at the 3′

end of the fragmented DNAs;

(3) connecting the end-filled DNAs having a 3′

end poly-adenine with sequencing linkers to obtain connected products;

(4) purifying the connected products to obtain the sequencing library;

wherein steps (1)-(3) are performed in a single reaction tube.

2. The method according to claim 1, characterized in that, there is no DNA purification steps among steps (1)-(3).

3. The method according to claim 1, characterized in that, the method does not comprise a step of PCR amplification.

4. The method according to claim 1, characterized in that, there is a step of inactivating the double-strand DNA fragmentation enzymes between the step (1) and step (2).

5. The method according to claim 1, characterized in that, the double-strand DNA fragmentation enzyme is an enzyme mixture of a nonspecific notch nuclease and a T7 endonuclease mutant.

6. The method according to claim 4, characterized in that, the nonspecific notch nuclease is a Vvn nuclease, and the T7 endonuclease mutant is a T7 endonuclease having a mutation or mutations in the bridging region of two catalytic domains.

7. The method according to claim 1, characterized in that, step (2) is carried out by T4 DNA polymerase and Taq enzyme.

8. The method according to claim 1, characterized in that, the sequencing linkers are double-strand sequencing linkers matched to sequencing platforms.

9. The method according to claim 1, characterized in that, step (3) is carried out by T4 DNA ligase.

10. The method according to claim 1, characterized in that, the sequencing library is suitable for the second generation high throughput sequencing platforms.

11. A kit for constructing a sequencing library for use in detecting chromosome copy number variation, characterized in that, the kit comprises:

double-strand DNA fragmentation enzymes and buffer I for DNA fragmentation, enzymes for DNA end-filling, enzymes for adding poly-adenine at 3′

end, sequencing linkers, ligase and buffer III for ligation, wherein the DNA fragmentation, DNA end-filling and addition of poly-adenine at 3′

end are carried out in a single reaction tube.

12. The kit according to claim 11, characterized in that, the double-strand DNA fragmentation enzyme is an enzyme mixture of a nonspecific notch nuclease and a T7 endonuclease mutant.

13. The kit according to claim 12, characterized in that, the nonspecific notch nuclease is a Vvn nuclease, and the T7 endonuclease mutant is a T7 endonuclease having a mutation or mutations in the bridging region of two catalytic domains.

14. The kit according to claim 11, characterized in that, the enzyme for DNA end-filling is T4 DNA polymerase, and the enzyme for adding poly-adenine at 3′

end is Taq enzyme.

15. The kit according to claim 11, characterized in that, the sequencing linkers are double-strand sequencing linkers matched to sequencing platforms.

16. The kit according to claim 11, characterized in that, the ligase is T4 DNA ligase.

17. The kit according to claim 11, characterized in that, it further comprises buffer II for inactivating the double-strand DNA fragmentation enzymes.