DNA FRAGMENT DETECTION METHOD, DNA FRAGMENT DETECTION KIT AND THE USE THEREOF

• US 20140234850A1
• Filed: 07/11/2012
• Published: 08/21/2014
• 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, Daixing, Gao, Yang, Tian, Feng, Zhou, Zhongchun, Xu, Genming
• Granted Patent: US 9,540,687 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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, wherein comprising the steps of:

designing primers according to a test site or a 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 the PCR amplification product.

2. The method according to claim 1, wherein the primers are a primer pair comprising of adjacent primers which extend backwards.

3. The method according to claim 2, wherein the primers of the primer pair which extend backwards are all located on the 5′

end or 3′

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

4. The method according to claim 2, 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.

5. The method according to claim 4, wherein the interval between the primers of the primer pair which extend backwards is 0-50 base pairs.

6. The method according to claim 5, wherein the interval between the primers of the primer pair which extend backwards is 0-10 base pairs.

7. The method according to claim 2, wherein the bases on the 5′

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

8. The method according to claim 1, wherein the primers are one primer.

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

10. The method according to claim 1, wherein the cyclization is CircLigase-mediated double-chain DNA cyclization.

11. 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.

12. 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.

13. A DNA fragment detection kit, wherein comprising:

a DNA extraction reagent, a DNA cyclase, target DNA amplification primers and an amplification reagent.

14. The DNA fragment detection kit according to claim 13, wherein the kit further comprising:

target DNA pre-amplification primers and a pre-amplification reagent.

15. The DNA fragment detection kit according to claim 13, wherein the target DNA amplification primers are a primer pair comprising of adjacent primers which extend backwards.

16. The DNA fragment detection kit according to claim 15, wherein the primers of the primer pair which extend backwards are all located on the 5′

end or 3′

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

17. The DNA fragment detection kit according to claim 16, wherein interval between the primers of the primer pair which extend backwards is 0-½

of the total base pairs of the DNA fragment.

22. The DNA fragment detection kit according to claim 13, wherein the test site or the test region of the DNA fragment comprise of insertion, deletion, substitution or fusion gene mutation.

23. The use of the DNA fragment detection kit according to claim 13 in detecting cancers, congenital monogenic diseases, chromosomal aberration diseases and infectious diseases.

24. The use of the DNA fragment detection kit according to claim 13 in vitro detection of DNA mutations.

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