Method and kit thereof for detecting BRAF gene mutation

A kit and gene technology, applied in the field of a method for detecting BRAF gene mutation and its kit, can solve the problems of melting temperature drop, lack of specificity, sensitivity and accuracy, etc.

Inactive Publication Date: 2015-09-23
李跃 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of this method are: 1) Each specific primer or probe can only be identified for a certain type of gene mutation; 2) False positives due to single base mismatches may occur
2) For mismatched PNA/DNA, even if there is only one base mismatch, its

Method used

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  • Method and kit thereof for detecting BRAF gene mutation
  • Method and kit thereof for detecting BRAF gene mutation
  • Method and kit thereof for detecting BRAF gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1 is directed at the detection of V600E and V600K mutant plasmid standards

[0083] V600E contains two nucleotide mutation sequences: GAG and GAA

[0084] Main source of reagents:

[0085] 1.1 Designing PNAs

[0086] PNA was synthesized by Panagene in South Korea and PNA Bio Inc. in the United States.

[0087] PNA-NBN-W:NH 2 -TTGGTCTAGCTACA NBN AAATC-COOH; wherein, N represents A, C, G or T, and B represents C, G or T; the described merged sequences are all mixed at a ratio of 1:1.

[0088] PNA-HNN-K: NH 2 -TTGGTCTAGCTACA HNN AAATC-COOH; where, H means A, C or T, N Indicates A, C, G or T; the described merged sequences are all mixed 1:1.

[0089] PNA-BNN-E1E2:NH 2 -TTGGTCTAGCTACA BNN AAATC-COOH; where,B means C, G or T, N Indicates A, C, G or T; the described merged sequences are all mixed 1:1.

[0090] 1.2 Design of Tagman MGB probe

[0091] The Tagman MGB probe was synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.

[0092] Probe 1 (...

Embodiment 2

[0138] Example 2 Detection of V600E and V600K mutations in colorectal cancer tissue samples

[0139] 2.1 Specimen collection:

[0140] Collect surgically resected fresh colorectal cancer tissue samples from 8 cases of cancer (the diagnosis of cancer is based on clinical and pathological diagnosis, including pathological diagnosis), and the tissue is about 1mg.

[0141] 2.2 DNA extraction method:

[0142]Select a commercially available Qiangen Genomic DNA Extraction Kit, perform genomic DNA extraction according to the instructions, dissolve the DNA with 100 μl TE, and store in -80°C in aliquots.

[0143] 2.3 DNA concentration determination

[0144] Concentrations were measured using a NanoDrop ND-1000 full-wavelength UV / visible scanning spectrophotometer.

[0145] 2.4 Specimens for V600E and V600K mutation detection

[0146] Take 125ng of sample DNA, and perform ABC three-tube detection according to method 1.6 in Example 1.

[0147] 2.5 Result Analysis

[0148] Refer to 1...

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Abstract

The invention discloses a method and kit thereof for detecting BARF gene mutation. The method comprises the following steps: (1) carrying out reactions in tube A, B, and C at the same time, carrying out BARF gene V600E and V600K mutation realtime quantitative PCR detection on a plasmid standard with a known mutation amount so as to obtain standard curves; (2) extracting and purifying the DNA of a sample, measuring the concentration, carrying out realtime quantitative PCR detection on the sample according to the tube A, B, and C reaction systems in the step (1), judging whether the gene mutation exists or not and the mutation amount according to the standard curves and the amplification fluorescence signals. The kit comprises a primer pair for detecting the No.15 exon mutation V600 E and V600K of BRAF gene, a probe, and a PNA repressing sequence. The provided method and kit can more precisely detect the No.15 exon mutation V600 E and V600K of BRAF gene, and the detection sensitivity is greatly improved.

Description

technical field [0001] The invention relates to a method for detecting gene mutation and its kit, in particular to a method for detecting BRAF gene mutation and its kit. Background technique [0002] The incidence of colorectal cancer is increasing year by year, and patients with advanced and postoperative recurrence need biological targeted therapy. The U.S. FDA and my country’s postoperative treatment plan for colorectal cancer clearly point out that when implementing first-line and second-line chemotherapy regimens, when it is planned to add targeted drugs targeting EGFR receptor antibodies (cetuximab and panitumumab), Drug susceptibility gene mutation detection is required. International large-sample multi-center studies have shown that simultaneous detection of KRAS, BRAF, NRAS, and PIK3CA gene mutations, followed by tandem diagnosis in this order, can maximize and accurately predict patients’ sensitivity to antibody-targeted drugs (see literature Roock WD, Claes B, Be...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6827C12Q1/6886C12Q2600/156C12Q2561/113C12Q2561/101C12Q2545/114C12Q2531/113
Inventor 李跃高小祐孙景凤
Owner 李跃
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