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Method for detecting BRAF (block repeat active flag) gene mutation accurately based on probe fusion technology

A melting and technical technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, fluorescence/phosphorescence, etc., can solve the problems of inaccurate detection and low sensitivity, and achieve low quality requirements, high sensitivity, and optimized amplification effect of effect

Active Publication Date: 2013-05-15
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 BRAF gene mutations based on 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 detecting BRAF (block repeat active flag) gene mutation accurately based on probe fusion technology
  • Method for detecting BRAF (block repeat active flag) gene mutation accurately based on probe fusion technology
  • Method for detecting BRAF (block repeat active flag) gene mutation accurately based on probe fusion technology

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Embodiment Construction

[0033] The present invention combines molecular beacon probes with extension-blocking primers, and uses a polymerase without 5'→3' exonuclease activity to rapidly detect BRAF 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 whose self-extension products can form a "neck loop" structure; in addition to the normal primer sequence, the 5' end of the extension-refractory primer contains an additional base sequence, which The segment 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 ring" structure; due to the difference in sequence between the wild-type and mutant templates, the formed "neck ring" "There is a significant difference in structural strength (Tm value).

[0035]The pre...

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Abstract

The invention provides a method for detecting BRAF (block repeat active flag) gene mutation accurately based on a probe fusion technology. Through the use of the method, not only consumable items are saved, but also characteristics of high sensitivity and specificity are realized and the detection result is more reliable. The method comprises the following steps of (1) design and synthesis of a molecular beacon probe and a template amplification primer, wherein a designed upstream primer (27nt) is 5'-TGTTTTCCTTTACTTACTACACCTCAG-3', an extension refractory primer (43nt) is 5'-acGCTACAGTGAAATCTCGGACAACTGTTCAAACTGATGGGAC-3', and the molecular beacon probe (35nt) is FAM-ccgcaTCGAGATTTCACTGTAGCTAGACCAtgcgg-BHQ2; (2) extraction of a genomic DNA (deoxyribonucleic acid) of a tested sample; (3) preparation of a reaction system; and (4) reaction of PCR (polymerase chain reaction).

Description

technical field [0001] The invention relates to a method for detecting BRAF gene mutation based on 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 BRAF 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 BRAF gene is activated after receiving upstream signal activation, and After the signal is transmitted to the downstream cytokines, the inactive state will be restored; while the protein encod...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 戴鹏高陈超刘金辉焦维丽邹晖
Owner 陕西佰美医学检验有限公司
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