Preparation of adapter-ligated amplicons

• US 10,648,031 B2
• Filed: 08/21/2015
• Issued: 05/12/2020
• 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
• Primary Examiner(s): Vivlemore, Tracy
• Assistant Examiner(s): Kaup, Sahana S
• Application Number:
  US15/508,251
• Publication Number:
  US 20170283869A1
• Time in Patent Office: 1,726 Days
• Field of Search: None
• 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, wherein the double-stranded amplicons are provided directly resulting from an amplification reaction without a subsequent chemical modification of the 3′ and

/or 5′

termini, and

(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, wherein said double-stranded amplicons are double-stranded PCR amplicons.

3. The method of claim 2, wherein said double-stranded PCR amplicons result from a PCR using a DNA polymerase comprising terminal adenylyltransferase activity.

4. The method of claim 3, wherein 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, wherein said DNA adapter molecules comprise a T overhang.

6. The method of claim 1, wherein said polynucleotide kinase is T4 polynucleotide kinase or T4 polynucleotide kinase (3′

phosphatase minus).

7. The method of claim 1, wherein 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, wherein said DNA adapter molecules are sequencing DNA adapter molecules.

9. The method of claim 1, wherein 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, further comprising, after step (ii),

(iii) isolating adapter-ligated PCR amplicons.

12. The method of claim 1, wherein said DNA adapter molecules are DNA adapter molecules for next generation sequencing (NGS).

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,

to obtain a reaction mixture, wherein the double-stranded amplicons are provided directly resulting from an amplification reaction without a subsequent chemical modification of the 3′ and

/or 5′

termini, and

(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.

13. The method of claim 11, wherein said DNA adapter molecules comprise a T-overhang.