PREPARATION OF ADAPTER-LIGATED AMPLICONS

• US 20170283869A1
• Filed: 08/21/2015
• Published: 10/05/2017
• Est. Priority Date: 09/05/2014
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Fang, Nan, Loeffert, Dirk
• Granted Patent: US 10,648,031 B2
• Time in Patent Office: Days
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/131 Terminal transferase
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

The present invention is directed to novel methods and kits to be employed for preparing adapter-ligated amplicons or a sequencing library of a target DNA, respectively.

Claims

1. A method of preparing adapter-ligated amplicons, comprising the steps of:

(i) contacting double-stranded amplicons with

at least one polynucleotide kinase,

at least one DNA ligase, and

DNA adapter molecules,

to obtain a reaction mixture,

(ii) incubating said reaction mixture under conditions simultaneously allowing

a 5′

phosphorylation of said double-stranded amplicons by said polynucleotide kinase, and

a joining of said DNA adapter molecules to at least one end of said double-stranded amplicons by said DNA ligase,

to obtain adapter-ligated amplicons.

2. The method of claim 1, characterized in that said double-stranded amplicons are double-stranded PCR amplicons.

3. The method of claim 2, characterized in that said double-stranded PCR amplicons resulting from a PCR using a DNA polymerase comprising terminal adenylyltransferase activity.

4. The method of claim 3, characterized in that said DNA polymerase is selected from the group consisting of:

Taq polymerase, Klenow fragment, TopTaq polymerase, Tfl-Polymerase, Tma-Polymerase, Tne-Polymerase and Tth-Polymerase.

5. The method of claim 1, characterized in that said DNA adapter molecules comprising a T overhang.

6. The method of claim 1, characterized in that said polynucleotide kinase is selected from the group consisting of:

T4 polynucleotide kinase, T4 polynucleotide kinase (3′

phosphatase minus).

7. The method of claim 1, characterized in that said DNA ligase is selected from the group consisting of:

T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, E. coli DNA Ligase, Taq DNA ligase, and 9°

N DNA ligase.

8. The method of claim 1, characterized in that said DNA adapter molecules are sequencing DNA adapter molecules, preferably DNA adapter molecules for next generation sequencing (NGS).

9. The method of claim 1, characterized in that in step (i) said contacting is realized by subjecting said double-stranded amplicons, said at least one polynucleotide kinase, said at least one DNA ligase, and said DNA adapter molecules to one single reaction container.

10. The method of claim 1, characterized in that after step (ii) the following step is performed:

(iii) isolating adapter-ligated PCR amplicons.

12. A kit for preparing adapter-ligated PCR amplicons, comprising

(i) at least one polynucleotide kinase,

(ii) at least one DNA ligase,

(iii) DNA adapter molecules, preferably comprising a T-overhang,

(vi) reaction buffer configured to simultaneously allowing the enzymatic functioning of said at least one polynucleotide kinase and said at least one DNA ligase, and

(v) a manual for performing the method according to claim 1.

13. A kit for preparing a sequencing library of a target DNA, comprising

(i) at least one DNA polymerase, preferably comprising terminal adenylyltransferase activity,

(ii) at least one polynucleotide kinase,

(iii) at least one DNA ligase,

(iv) DNA adapter molecules, preferably comprising a T-overhang,

(v) PCR buffer,

(vi) reaction buffer configured to simultaneously allowing the enzymatic functioning of said at least one polynucleotide kinase and said at least one DNA ligase, and

(vi) a manual for performing the method according to claim 9.

14. The kit of claim 13, characterized in that said DNA polymerase is a Taq polymerase.

15. The kit of claim 12, characterized in that said polynucleotide kinase is selected from the group consisting of:

T4 polynucleotide kinase, Klenow fragment, TopTaq polymerase, Tfl-Polymerase, Tma-Polymerase, Tne-Polymerase and Tth-Polymerase, preferably said DNA ligase is selected from the group consisting of;

T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, E. coli DNA Ligase, Taq DNA ligase, and 9°

N DNA ligase.

16. The kit of claim 13, characterized in that said polynucleotide kinase is selected from the group consisting of:

T4 polynucleotide kinase, Klenow fragment, TopTaq polymerase, Tfl-Polymerase, Tma-Polymerase, Tne-Polymerase and Tth-Polymerase, preferably said DNA ligase is selected from the group consisting of;

T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, E. coli DNA Ligase, Taq DNA ligase, and 9°

N DNA ligase.

11. A method of preparing a sequencing library of a target DNA, comprising the steps of:

(i) subjecting said target DNA to a PCR under conditions resulting in double-stranded PCR amplicons of said target DNA,

(ii) contacting said double-stranded PCR amplicons with

at least one polynucleotide kinase,

at least one DNA ligase, and

DNA adapter molecules, preferably comprising a T-overhang,

to obtain a reaction mixture,

(iii) incubating said reaction mixture under conditions simultaneously allowing

a 5′

phosphorylation of said double-stranded PCR amplicons by said DNA ligase, and

a joining of said DNA adapter molecules to at least one end of said double-stranded PCR amplicons by said DNA ligase,

to obtain a sequencing library of said target DNA.