Method for accurately detecting KRAS gene mutation by use of probe melting technology

A melting and technical technology, applied in the field of KRAS gene mutation detection, can solve the problems of low sensitivity and inaccurate detection, and achieve the effects of high sensitivity, improved detection efficiency, and easy analysis of experimental results.

Active Publication Date: 2014-11-05
陕西佰美医学检验有限公司
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Problems solved by technology

[0005] In order to overcome the shortcomings of low sensitivity and inaccurate detection of the traditional probe melting curve method, the present invention proposes a method for accurately detecting KRAS gene mutations using probe melting technology, which not only saves consumables, but also has the characteristics of high sensitivity and high specificity , the test results are more reliable

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  • Method for accurately detecting KRAS gene mutation by use of probe melting technology
  • Method for accurately detecting KRAS gene mutation by use of probe melting technology
  • Method for accurately detecting KRAS gene mutation by use of probe melting technology

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Embodiment Construction

[0033] The present invention combines molecular beacon probes with extension-blocking primers, and utilizes a polymerase without 5'→3' exonuclease activity to rapidly detect KRAS mutations in vitro, which not only saves consumables, but also has high sensitivity and high specificity The characteristics make the test results more reliable.

[0034] Extension-Refractory primers (Extension-Refractory primers) are primers that can form a "neck loop" structure with their own extension products; in addition to the normal primer sequence, the 5' end of the extension-refractory primer contains an additional base sequence, This sequence can be complementary to a base sequence including the mutation site in the single strand of the product obtained by its own extension, forming a "neck loop" structure; due to the difference in sequence between the wild-type and mutant templates, the formed "neck loop" There is a significant difference in the strength (Tm value) of the "ring" structure. ...

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Abstract

The invention provides a method for accurately detecting KRAS gene mutation by use of probe melting technology, which saves consumable materials and has the characteristics of high sensitivity and high specificity and obtains a more reliable detection result. The method comprises the following steps of: (1) designing and synthesizing a molecular beacon probe and template amplification primers, wherein the designed forward primer (22nt) is 5'-CAGACTGTGTTTCTCCCTTCTC-3', the extension-refractory primer (36n6) is 5'-taAGGTCAAGAGGACTCATGTACTGGTCCCTCATTG-3', and the molecular beacon probe (34nt) is FAM-acgcATTGTACTCCTCTTGACCTGCTGTatgcgt-BHQ2; (2) extracting the genomic DNA of the detected sample; (3) preparing a reaction system; and (4) performing PCR reaction.

Description

technical field [0001] The invention relates to a method for detecting KRAS gene mutation by using probe melting technology. Background technique [0002] Gene mutation refers to the change of base composition or arrangement sequence in the structure and function of genomic DNA molecules, mainly including base substitution and fragment insertion and deletion, and is one of the important causes of genotype diseases. Gene mutation analysis plays a very important role in biomedical research, especially in genotypic disease diagnosis and pathological research. The protein encoded by the KRAS gene is involved in the cell signal transduction pathway mediated by the epidermal growth factor receptor (EGFR), affecting cell proliferation, growth and metastasis; the protein encoded by the normal KRAS gene is activated after receiving upstream signal activation, and The inactivated state will be restored after the signal is transmitted to the downstream cytokines; while the protein enc...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 陈超戴鹏高刘金辉宿志瑞邹晖
Owner 陕西佰美医学检验有限公司
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