DNA fragment detection method, DNA fragment detection kit and the use thereof

• US 9,540,687 B2
• Filed: 07/11/2012
• Issued: 01/10/2017
• Est. Priority Date: 07/11/2012
• Status: Active Grant

• Current Assignee: Berry Genomics Co., Ltd.
• Original Assignee: Berry Genomics Co., Ltd.
• Inventors: Zhang, Jianguang, Zhou, Zhongchun, Zhou, Daixing, Xu, Genming, Gao, Yang, Tian, Feng
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US14/347,113
• Publication Number:
  US 20140234850A1
• Time in Patent Office: 1,644 Days
• Field of Search: 435/91.2
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides

Abstract

The disclosure claims a cleaved Deoxyribonucleic acid (DNA) detection method, a DNA fragment detection kit and use thereof. Wherein, the method includes the steps of: designing primers according to a test site or a test region of the DNA fragment; cyclizing the DNA fragment to obtain acyclized DNA; implementing Polymerase Chain Reaction (PCR) amplification for the cyclized DNA by using the primers; and detecting the PCR amplification product. In the disclosure, by cyclizing the DNA fragment, the amplification can be implemented even if only one PCR primer can match with a template, thus, the adaption range and effective template amount of the primer amplification can be greatly increased, and the detection sensitivity of the DNA fragment can be greatly improved.

Claims

1. A DNA fragment detection method, comprising the steps of:

designing primers based on a test site adjacent sequence or a test region adjacent sequence of a DNA fragment, wherein the primers are a primer pair comprising of adjacent primers which extend backwards and the primers of the primer pair which extend backwards are all located on the 5′

end 3′

end of the test site or the test region of the DNA fragment;

cyclizing the DNA fragment to obtain a cyclized DNA;

implementing PCR amplification for the cyclized DNA by using the primers; and

detecting a product of the PCR amplification,

wherein the DNA fragment refers to a short fragment DNA which is formed by natural random fragmentation of biological genomic DNA, and has a length of 20 bp to 2000 bp.

2. The method according to claim 1, wherein the interval between the primers of the primer pair which extend backwards is 0-½

of the total base pairs of the DNA fragment.

3. The method according to claim 2, wherein the interval between the primers of the primer pair which extend backwards is 0-50 base pairs.

4. The method according to claim 3, wherein the interval between the primers of the primer pair which extend backwards is 0-10 base pairs.

5. The method according to claim 1, wherein the bases on the 5′

end of the primers of the primer pair which extend backwards are overlapped partly.

6. The method according to claim 1, wherein before cyclizing the DNA fragment, the method further includes a step of pre-amplifying the DNA fragment.

7. The method according to claim 1, wherein the DNA fragment comprises of the plasma free DNA, urine free DNA, sweat free DNA, saliva free DNA, or the DNA extracted from formalin-fixed and paraffin-embedded tissues.

8. The method according to claim 1, wherein the test site or the test region of the DNA fragment include insertion, deletion, substitution or fusion gene mutation.

9. A DNA fragment detection method, comprising the steps of:

designing one primer according to a test site adjacent sequence or a test region adjacent sequence of the DNA fragment, the primer located on the 5′

end or 3′

end of the test site or the test region of the DNA fragment;

cyclizing the DNA fragment to obtain a cyclized DNA;

implementing PCR amplification for the cyclized DNA by using the primer; and

detecting the PCR amplification product, the DNA fragment refers to a short fragment DNA which is formed by natural random fragmentation of biological genomic DNA, and has the length of 20-2000 bp.