Method of amplifying telomere

Patent metadata

Field	Value
Publication number	US-10059985-B2
Application number	US-201514866386-A
Country	US
Kind code	B2
Filing date	Sep 25, 2015
Priority date	Sep 26, 2014
Publication date	Aug 28, 2018
Grant date	Aug 28, 2018

Abstract

A method of amplifying a telomere of genomic DNA using an adaptor sequence, and a composition and a kit for amplifying the telomere of genomic DNA.

First claim

What is claimed is:

1. A method of amplifying a telomere of genomic deoxyribonucleic acid (DNA), the method comprising:
preparing a telomere fragment by incubating a sample comprising genomic DNA including a telomere with at least one nuclease, wherein the nuclease is a restriction enzyme and cleaves the genomic DNA to produce a telomere fragment having 3′-overhang;
preparing a ligated telomere fragment by incubating the telomere fragment with at least one single-stranded adaptor nucleic acid and a ligase, wherein the at least one single-stranded adaptor nucleic acid comprises a first region located at the 3′ end of the single stranded adaptor nucleic acid that is complementary to a nucleic acid sequence on the 3′ overhang of the telomere fragment, thereby forming an annealed product between the telomere fragment and the single-stranded adaptor nucleic acid, and ligating the telomere fragment to the single-stranded adaptor nucleic acid in the annealed product to form the ligated telomere;
preparing a blunt-end ligated telomere fragment by incubating the ligated telomere fragment with a single-strand specific exonuclease that cleaves in the 3′ to 5′ direction, a first nucleic acid polymerase, or a combination thereof;
preparing a circular telomere fragment by incubating the blunt-end ligated telomere fragment with a ligase, thereby ligating the 5′ end of the one strand with the 3′ end of the same strand and the 3′ end of the other strand with the 5′ end of the same strand; and
amplifying the telomere fragment by incubating the circular telomere fragment with a forward primer, a reverse primer, or a combination thereof, and a second nucleic acid polymerase,
wherein the forward primer comprises a nucleic acid sequence identical to at least two contiguous nucleotides located 5′ of the first region of the single-stranded adaptor nucleic acid and the reverse primer comprises a nucleic acid sequence complementary to the at least two contiguous nucleotides located 5′ of the first region of the single-stranded adaptor nucleic acid.

2. The method of claim 1 , wherein the sample is a biological sample.

3. The method of claim 1 , wherein the single-stranded adaptor nucleic acid further comprises a second region comprising a nucleic acid sequence identical to the nucleic acid sequence of the forward primer, and a third region comprising a nucleic acid sequence complementary to the reverse primer, from a 3′-end of the adaptor nucleic acid.

4. The method of claim 1 , wherein the first region comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 2 to 7.

5. The method of claim 1 , wherein the exonuclease is Exonuclease I.

6. The method of claim 1 , wherein the first nucleic acid polymerase and/or the second nucleic acid polymerase is a DNA polymerase.

7. The method of claim 1 , further comprising phosphorylating a 5′-end of the ligated telomere fragment comprising a blunt end by incubating said ligated telomere fragment comprising a blunt end with a polynucleotide kinase (PNK).

8. The method of claim 1 , wherein amplifying the telomere fragment is performed by a thermal cycling amplification method or an isothermal amplification method.

9. The method of claim 1 , wherein the amplifying of the telomere fragment is performed by a polymerase chain reaction (PCR), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), ligase chain reaction (LCR), strand displacement amplification (SDA), rolling circle amplification (RCA), or a combination thereof.

Assignees

• Samsung Electronics Co Ltd

Inventors

• Choi Kobong
• Kang Hyuntae
• Keum Jungwon

Classifications

• C12Q2521/501

  Ligase · CPC title

• C12Q2531/131

  Inverse PCR · CPC title

• C12Q1/6862 Primary

  Ligase chain reaction [LCR] · CPC title

• C12Q2521/319

  Exonuclease · CPC title

• C12Q1/6855 Primary

  Ligating adaptors · CPC title