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Method and kit for detecting BRAF (v-raf mourine sarcoma viral oncogene homolog B1) gene mutation

A kit and gene technology, applied in the field of detecting BRAF gene mutation using pyrosequencing technology, can solve the problems of limited detection sensitivity and specificity of the kit, and achieve the effects of low detection cost, simple operation and high specificity

Active Publication Date: 2013-12-18
北京明谛生物医药科技有限公司
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Problems solved by technology

[0008] For example, the Chinese invention patent with the publication number CN102925555A discloses a pair of sequencing primers and a kit for qualitatively detecting human BRAFV600E gene mutations using pyrosequencing technology. The kit includes uracil DNA glycosylase, Taq polymerase, PCR reaction solution, PCR amplification primers, pyrosequencing primers, and positive controls, however, the detection sensitivity and specificity of this kit are limited

Method used

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  • Method and kit for detecting BRAF (v-raf mourine sarcoma viral oncogene homolog B1) gene mutation
  • Method and kit for detecting BRAF (v-raf mourine sarcoma viral oncogene homolog B1) gene mutation
  • Method and kit for detecting BRAF (v-raf mourine sarcoma viral oncogene homolog B1) gene mutation

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Experimental program
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Embodiment 1B

[0053] The investigation of embodiment 1 BRAF gene mutation detection method

[0054] 1. Construction of wild type plasmid and mutant plasmid

[0055] The vector used for plasmid construction is the pMD18-T vector, and its insert sequence is the sequence fragment of the 171241-171660 position of the BRAF gene (NCBI Reference Sequence: NG_007873), in which the wild-type plasmid contains the T sequence of the 1799 position of the BRAF gene, and the mutant type The plasmid contains the 1799A sequence of the BRAF gene; the construction of the plasmid was completed by TaKaRa Company;

[0056] Transform the constructed wild-type plasmid and mutant-type plasmid into Escherichia coli respectively, and after culturing, save the corresponding bacterial liquid;

[0057] 2. Extraction of plasmid

[0058] Use the QIAGEN Plasmid Mini Kit to extract wild-type plasmids and mutant plasmids from the cultivated bacterial solution containing the above-mentioned wild-type plasmids and mutant pla...

Embodiment 2

[0074] Example 2 Detection of formalin-fixed paraffin-embedded human tumor tissue samples

[0075] Genomic DNA was extracted from formalin-fixed paraffin-embedded human tumor tissue samples 1-3 using QIAamp DNA FFPE Tissue Kit respectively, and the specific operation steps were referred to the kit instructions to obtain DNA extraction solutions 1-3;

[0076] According to the method described in Example 1, the DNA extracts 1-3 were respectively subjected to BRAF gene amplification (that is, the DNA extract was used to replace the plasmid in the amplification system), the preparation of single-stranded DNA, and the pyrosequencing reaction. The result is as Figure 6-8 As shown; the instrument according to the electropherogram judgment results show that: the BRAF gene 1799 site of sample 1 is pure and wild type (100% T base); the BRAF gene 1799 site of sample 2 is heterozygous mutant (13% A base base); the 1799 site of the BRAF gene in sample 3 was a heterozygous mutant (42% A b...

Embodiment 3

[0077] Example 3 Detection of Human Frozen Tumor Tissue Samples

[0078] Use the QIAamp DNA FFPE Tissue Kit to extract genomic DNA from human frozen tumor tissue samples 4-6, and refer to the kit instructions for specific operations to obtain DNA extraction solutions 4-6;

[0079] According to the method described in Example 1, BRAF gene amplification, single-stranded DNA preparation and pyrosequencing reaction were respectively performed on the DNA extract 4-6, and the sequencing results were as follows Figure 9-11 As shown; the instrument according to the electropherogram judgment results show that: the 1799 site of the BRAF gene in sample 4 is pure and wild type (100% T base); the 1799 site of the BRAF gene in sample 5 is a heterozygous mutant (21% A base base); the 1799 site of the BRAF gene in sample 6 was a heterozygous mutant (85% A base).

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Abstract

The invention discloses a primer, a kit and a method for detecting BRAF (v-raf mourine sarcoma viral oncogene homolog B1) gene mutation. The primer for detecting the BRAF gene mutation comprises a forward amplification primer with a base sequence represented by SEQ ID NO:1, a backward amplification primer with a base sequence represented by SEQ ID NO:2 and a sequencing primer with a base sequence represented by SEQ ID NO:3, wherein the 5' tail end of the forward amplification primer and / or the backward amplification primer is provided with a biotin label. The method and the kit for detecting the BRAF gene mutation have the advantages of simplicity and rapidness for operation, high flux, low detection cost and the like, especially have good specificity, high sensitivity and high accuracy and have wide application prospect.

Description

technical field [0001] The invention relates to a method for detecting BRAF gene mutation, in particular to a method for detecting BRAF gene mutation by pyrosequencing technology and a kit thereof. Background technique [0002] Murine sarcoma viral oncogene homolog B1 (v-raf moourine sarcoma viral oncogene homolog B1, BRAF), a member of the RAF family, is located on chromosome 7q34, with a size of 190kb, containing seven transcribed regions, including 18 exogenous exon, encoding a 67KD-99KD serine / threonine protein kinase, which transduces signals from RAS to MEK1 / 2, thereby participating in cell proliferation, differentiation and apoptosis (Ikenoue T, CancerRes, 2003, 63( 23): 8132-37). Studies have shown that there are two main mutations in BRAF. 89% of the mutations occur in the activation region on exon 15, and about 92% of the mutations are located at the 1799th nucleotide (T mutation to A), resulting in its protein product The valine in residue 600 was replaced by gl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 王殊刘建云
Owner 北京明谛生物医药科技有限公司
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