# US 2014/0234850 A1 · Dna fragment detection method, dna fragment detection kit and the use thereof

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- Number: US 2014/0234850 A1
- Document ID: US20140234850A1
- Status: Published application
- Kind: A1
- Application number: 14/347,113
- Filed: 2012-07-11
- Published: 2014-08-21
- Earliest claimed: 2012-07-11
- Assignees: Berry Genomics Co., Ltd.
- Applicants: Berry Genomics Co., Ltd.
- Inventors: Jianguang Zhang; Zhongchun Zhou; Daixing Zhou; Genming Xu; Yang Gao; Feng Tian
- Web page: https://patentgrep.com/patent/US20140234850A1

## CPC

- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 2)

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.

### Claim 4 (depends on 2)

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.

### Claim 5 (depends on 4)

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

### Claim 6 (depends on 5)

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

### Claim 7 (depends on 2)

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.

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

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.

### Claim 12 (depends on 1)

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.

### Claim 13

A DNA fragment detection kit, wherein comprising: a DNA extraction reagent, a DNA cyclase, target DNA amplification primers and an amplification reagent.

### Claim 14 (depends on 13)

The DNA fragment detection kit according to claim 13, wherein the kit further comprising: target DNA pre-amplification primers and a pre-amplification reagent.

### Claim 15 (depends on 13)

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.

### Claim 16 (depends on 15)

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.

### Claim 17 (depends on 16)

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.

### Claim 18

(canceled)

### Claim 19

(canceled)

### Claim 20

(canceled)

### Claim 21

(canceled)

### Claim 22 (depends on 13)

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.

### Claim 23 (depends on 13)

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

### Claim 24 (depends on 13)

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