New method for KRAS gene mutation detection

A new method, a technology for connecting probes, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of poor specificity, low sensitivity, and high cost of mutation detection, achieving low cost and high sensitivity , Simple and convenient operation

Inactive Publication Date: 2018-12-14
ZUNYI MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a KRAS mutati

Method used

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  • New method for KRAS gene mutation detection
  • New method for KRAS gene mutation detection
  • New method for KRAS gene mutation detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Construction of fluorescent biosensors and detection of KRAS mutations

[0019] 1. Materials and methods

[0020] 1.1 Materials

[0021] Taq DNA ligase and Bst DNA polymerase were purchased from NEB (Beijing) Co., Ltd. deoxynucleotide solution mixture (dNTPs) was purchased from Bao Biological Engineering (Dalian) Co., Ltd. Nuclease-free Water was purchased from Thermo Fisher Scientific (China) Co., Ltd. Paraffin-embedded tissue DNA rapid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The DNA strands purified by HPLC were synthesized and modified by Sangon Bioengineering (Shanghai) Co., Ltd. The clinical specimens used in the experiment came from the First Affiliated Hospital of Chongqing Medical University.

[0022] 1.2 Testing instruments

[0023] The ligation reaction was completed using a BIO-RAD PCR instrument, and the real-time fluorescence intensity was monitored by an Applied Biosystems real-time fluorescent quantitativ...

Embodiment 2

[0040] Validation of the feasibility of a ligation-based loop-mediated isothermal amplification method for detecting KRAS mutations

[0041] 1. Verification of the generation of dumbbell-shaped templates in ligation reactions

[0042] The connection product obtained in Example 1 is analyzed and verified by PAGE electrophoresis and fluorescence method, such as figure 1 The electrophoresis band in A shows that the ligation can only be performed under the conditions of the simultaneous presence of SLS1 (ligation probe 1), SLS2 (ligation probe 2), target sequence (MutDNA) and Taq ligase, resulting in a nucleic acid chain of about 110bp bring. figure 1 It can also be seen in B that the connection probe can be connected only in the presence of MutDNA, and the fluorescent groups and quenching groups respectively marked on the two connection probes are quenched due to the closer distance. Produces a lower fluorescent signal.

[0043] Such as figure 1 A: Band 1 is the DNA Marker of 5...

Embodiment 3

[0047] Fluorescence Biosensor and Optimization of Its Application Conditions

[0048] We also optimized the important conditions in the experimental process, namely, the mismatched base sites, the temperature of the ligation reaction, the number of cycles of the ligation reaction, the amount of Bst enzyme, and the temperature of the loop-mediated isothermal amplification. An optimized condition selected blank, WtDNA and different concentrations of MutDNA for a series of experiments.

[0049] 1. In order to investigate the influence of the mismatching sites in the junction probe SLS2 on the results of the fluorescent sensor with KRAS mutation, different mismatching sites were used in this experiment to construct the fluorescent sensor. The ratio of MutDNA to WtDNA POI (time corresponding to the point with the largest slope of the real-time fluorescence curve, the lower the POI value, the higher the amplification efficiency.) varies with different mismatched sites. When the mism...

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Abstract

A new method for KRAS gene mutation detection by means of loop-mediated isothermal amplification through ligation mediation includes steps of: (1) a ligation reaction for preparing a dumbbell-like template: adding ligation probe, ligase and a KRAS mutation sequence to a buffer liquid of a ligase, and placing the liquid in a PCR device for ligation, thus obtaining the dumbbell-like template througha reaction; (2) denaturing the dumbbell-like template with a loop-mediated isothermal amplification system, adding a stain and Bst enzyme, uniformly mixing the components, and performing a reaction in a real-time fluorescence PCR device, so that the primer can recognize the dumbbell-like template and trigger the loop-mediated isothermal amplification, and monitoring a fluorescent signal in real time. In the invention, the fluorescence analysis method for monitoring the KRAS mutation in practical samples is successfully constructed. The method for detecting the genome DNA extracted from the KRAS mutation samples has advantages of high sensitivity, specificity and anti-interference capability.

Description

technical field [0001] The invention relates to the technical field of mutation detection, in particular to a new method for detection of KRAS gene mutation. Background technique [0002] Colorectal cancer (CRC) is a common malignant tumor in the gastrointestinal tract. According to the statistics of the World Health Organization (WHO), there are about 1.2 million new cases of colorectal cancer each year in the world, and the annual death toll reaches more than 600,000. With the advent of targeted therapy drugs epidermal growth factor receptor inhibitor (EGFRI) cetuximab and panitumumab, the treatment of colorectal cancer has entered the era of targeted therapy. The KRAS gene is an important predictive biomarker that affects the responsiveness of tumor cells to EGFRI. KRAS gene is closely related to CRC, and the mutation rate in CRC is 35%-45%. Mutations in the KRAS gene will lead to continuous activation of the epidermal growth factor receptor (EGFR)-dependent RAS-RAF-MAP...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 闵迅付怡欣丁世家黄健周晓燕段晓雷袁建波
Owner ZUNYI MEDICAL UNIVERSITY
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