Kit and method for detecting PIK3CA gene mutation in plasma free DNA

A kit and plasma technology, applied in the field of molecular biology, can solve the problems of low detection throughput, loss of original information, complex library construction technology, etc., and achieve the effect of improving sensitivity and reducing sample usage.

Inactive Publication Date: 2017-10-17
CHENZHOU NO 1 PEOPLES HOSPITAL
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  • Claims
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Problems solved by technology

However, the NGS library construction technology is complex, and the conventional library construction sequencing method makes the tumor signal with a very low content in the plasma completely submerged in the background noise, and the original information

Method used

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  • Kit and method for detecting PIK3CA gene mutation in plasma free DNA

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Experimental program
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Embodiment Construction

[0028] Genotype Transformation of PIK3CA Gene E545K(G>A) Point Mutation

[0029] (1) PCR method is used to amplify the fragment to be identified at the E545K mutation site in the sample template, and the primers are as follows:

[0030] PIK3CA-F: 5'-AATCCAGAGGGGAAAAATATG-3' (SEQ ID NO.1)

[0031] PIK3CA-R: 5'-ATTTTAGCACTTACCTGTGACTC-3' (SEQ ID NO. 2).

[0032] (2) PCR reaction conditions of restriction endonuclease TspRI recognition site:

[0033] 1) The PCR system is shown in Table 1:

[0034] Table 1

[0035] HotStarTaq Plus DNA

0.3μL

10x PCR buffer

2.5μL

dNTP (each 10mM)

0.5μL

Upstream primer (10μM)

0.4μL

Downstream primer (10μM)

0.4μL

DNA template

2μL

ddH2O

Up to 25μL

[0036] Reaction program: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 sec, annealing at 52°C for 30 sec, extension at 72°C for 30 sec, 15 cycles;

[0037] 2) Add 0.8 μL of restriction endonuclease...

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Abstract

The invention relates to a kit and a method for detecting PIK3CA gene mutation in plasma free DNA. The kit contains specific primer pairs and high temperature-resistant restriction endonucleases; a specific primer comprises a sense primer shown as SEQ ID NO.1 and an antisense primer shown as SEQ ID NO.2; the high temperature-resistant restriction endonucleases are TspRI endonucleases. When the kit and the method for detecting the PIK3CA gene mutation, provided by the invention, are adopted for detecting the PIK3CA gene mutation, the sensitivity of mutation detection can be improved, the sample usage amount is reduced, and the clinical use and popularization are facilitated.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a kit and a method for detecting PIK3CA gene mutation in plasma free DNA by using PCR amplification detection combined with high-temperature-resistant restriction endonuclease treatment. Background technique [0002] The PIK3CA gene was detected by Volinia in 1994 using in situ hybridization. It is located at 3q26.3, 34kb in length, contains 21 exons, and encodes 1068 amino acids. This group of amino acids produces a group of 124kD proteins. PIK3CA encodes the p110 catalytic subunit of class I phosphatidylinositol-3-kinases (phosphatidylino-sitol 3-kinases, PI3Ks), namely PI3K p110a. [0003] Studies have found that PIK3CA is an oncogene. Under physiological conditions, the gene PIK3CA is expressed in normal tissues such as brain, lung, breast, gastrointestinal, cervix, and ovary, and has many important physiological functions such as regulating somatic cell proliferation, differen...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q1/6886C12Q2600/156C12Q2535/101C12Q2531/113C12Q2521/301
Inventor 罗迪贤蒲汪旸肖莉罗伟濠刑春根刘璇王凤娇胡政贺荣章李佳段丽丽张佳李凯
Owner CHENZHOU NO 1 PEOPLES HOSPITAL
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