Method for performing in-vitro accurate test on KRAS gene mutation

An accurate and genetic technology, applied in the field of detection of KRAS gene mutation, can solve the problems of insufficient specificity and achieve the effect of strong specificity, high sensitivity and high sensitivity

Active Publication Date: 2012-11-28
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 the traditional allele-specific PCR method, such as insufficient specificity and false positive phenomenon, the present invention provides a method for accurately detecting KRAS gene mutations in vitro. Accurate detection of KRAS mutation in vitro by PCR method

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  • Method for performing in-vitro accurate test on KRAS gene mutation
  • Method for performing in-vitro accurate test on KRAS gene mutation
  • Method for performing in-vitro accurate test on KRAS gene mutation

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Embodiment Construction

[0042] The invention is a technique for detecting KRAS gene mutation by using fluorescent allele-specific PCR and extension blocking primers, which is applied to in vitro KRAS gene sequence analysis and mutation detection.

[0043] 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.

[0044] The present invention designs allele-specific primers for seven typical mutations of the second exon of the K...

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Abstract

The invention provides a method for performing in-vitro accurate test on KRAS gene mutation, and the method is used for overcoming the defects that the conventional allele-specific polymerase chain reaction (PCR) method has insufficient specificity and false positive phenomenon. The method for performing the in-vitro accurate test on the KRAS gene mutation comprises the following steps of: (1) designing and synthetizing allele specific primers which comprise the allele specific primers espectively designed for 7 types of mutation of a typical KRAS gene exon 2 and are utilized as forward primers, wherein extension retardant primers are utilized as common downstream primers; (2) extracting the KRAS genomic deoxyribose nucleic acid (DNA) of a test sample, and additionally preparing the wild type genomic DNA (as wild type template); and (3) carrying out the real-time fluorescent PCR reaction. The test method has the advantages of strong specificity, high sensitivity, material conservation and time-saving property.

Description

technical field [0001] The invention relates to a method for detecting KRAS gene mutation. 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 encoded by the mutated KRAS gene is a...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 刘金辉陈超戴鹏高王鸿
Owner 陕西佰美医学检验有限公司
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