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Method and kit for enriching circulating tumor DNA on basis of multiplex PCR

A kit and tumor technology, applied in the direction of tumor/cancer cells, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of sample loss, sensitivity reduction, etc., to reduce errors, reduce false positives, and reduce costs. loss effect

Active Publication Date: 2019-08-13
常州桐树生物科技有限公司
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Problems solved by technology

However, most of the current detection processes will have a magnetic bead purification step in the middle, and each purification step will inevitably lead to sample loss, resulting in a decrease in detection sensitivity

Method used

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  • Method and kit for enriching circulating tumor DNA on basis of multiplex PCR
  • Method and kit for enriching circulating tumor DNA on basis of multiplex PCR
  • Method and kit for enriching circulating tumor DNA on basis of multiplex PCR

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Embodiment 1

[0096] The method of the invention is used to detect ctDNA tumor mutations in blood samples.

[0097] Specific steps are as follows:

[0098] 1. Test template preparation.

[0099] The cultured SK cell line and HCC1975 (containing EGFR: p.L858R mutation) cell line were subjected to DNA extraction according to the phenol-chloroform method, and then DNA fragmentation was performed with fragmentation enzyme from NEB Company. Then the DNA of the HCC1975 sample was incorporated into the SK sample at a ratio of 1%. The EGFR:p.L858R mutation frequency was quantified by ddPCR to obtain samples containing the EGFR:p.L858R mutation with known mutation frequency.

[0100] 2. Primer design. This embodiment designs 2 kinds of PCR primers

[0101] Primer I: PCR primer without U base.

[0102] Primer II: PCR primer with U base.

[0103] The length of the designed primers is 60-70bp, the formed primer-dimer sequence is between 70-100, the length of the designed universal sequencing adap...

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Abstract

The invention discloses a method and kit for enriching circulating tumor DNA on the basis of multiplex PCR. The method comprises the steps of (1), designing multiple PCR primer pairs, wherein PCR primers comprise an amplifying primer sequence used for amplifying a gene target region and a primer ligation sequence, and the primer ligation sequence is used for being linked with a universal sequencing linker of a sequencing platform; (2), taking a template containing the circulating tumor DNA and making the template and the PCR primer pairs in step (1) subjected to a first PCR reaction to obtaina first PCR reaction product; (3), adding a combined enzyme into the first PCR reaction product to digest residual PCR primers; (4), adding the universal sequencing linker into the first PCR reactionproduct and carrying out a second PCR reaction to obtain a sequencing library. The method and the kit have the effect of reducing sample loss.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and kit for enriching circulating tumor DNA based on multiplex PCR. Background technique [0002] With the completion of the human genome completion map, human beings have entered the post-genome era. In the post-genome era, it is first required to use the DNA sequence information of the human genome to study genes and their variations in order to detect the functional changes brought about by the variations. [0003] Usually, the occurrence and development of tumors involves the occurrence and development of multiple genes, and the research on the change of a single gene has shown great limitations. Only by comprehensively detecting the base mutations of multiple genes can effective monitoring of MRD, medication guidance, therapeutic efficacy and surgical procedures be carried out. Post-treatment etc. [0004] In recent years, extensive research has been carried out on th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12Q1/686
CPCC12N5/0693C12Q1/686C12Q2537/143
Inventor 蔡微菁徐泽
Owner 常州桐树生物科技有限公司
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