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K-Ras gene mutation detection method and applications thereof

A k-ras, human detection technology, applied in biochemical equipment and methods, microbial assay/inspection, DNA/RNA fragments, etc., can solve the problem of decreasing, increasing primer or probe melting temperature, base mismatch sensitivity, etc. question

Inactive Publication Date: 2018-06-29
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Locked nucleic acid (LNA-Locked nucleic acid) is a new type of nucleic acid analogue discovered in recent years. In the structure, the 2'-O and 4'-C positions of β-D-ribofuranose form a rigid structure through shrinkage so that LNA and DNA / The complex combined with RNA strands has high thermal stability and affinity. The incorporation of LNA can increase the melting temperature of primers or probes (the Tm value can be increased by 3-8°C), but it is also sensitive to base mismatches. Base mismatches can significantly reduce the Tm value

Method used

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  • K-Ras gene mutation detection method and applications thereof
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  • K-Ras gene mutation detection method and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: K-RAS gene amplification primer screening

[0044] (1) Reagent

[0045] name

company

place of origin

Agarose

Shanghai Sangon Bioengineering Co., Ltd.

Shanghai

100bp DNA Ladder

Tiangen Biochemical Technology Co., Ltd.

Beijing

UltraSYBR Mixture

Kangwei Century Biotechnology Co., Ltd.

Beijing

[0046] (2) Primer design and synthesis: Multiple pairs of K-RAS gene amplification primers were designed using "primer-blast" and sent to Sangon Bioengineering (Shanghai) Co., Ltd. The specific primer sequences are as follows:

[0047]

[0048]

[0049] (3) Real-time fluorescent quantitative PCR analysis

[0050] 20μl system, the ratio is as follows:

[0051]

[0052] The QPCR reaction procedure is as follows:

[0053]

[0054] For the results of melting curve analysis and DNA electrophoresis, see figure 1 and figure 2 , figure 1 The melting curve drawn for the product after real-tim...

Embodiment 2

[0055] Example 2 Real-time fluorescence quantitative PCR detects the sealing effect of LNA-Blocker

[0056] (1) Reagent

[0057]

[0058] (2)MgCl 2 Concentration optimization

[0059] Use Primer 12 primers to Roche (Roche) HRM reagent "LightCycler 480highresolution melting master" on the LightCycler 480 (Roche) machine to optimize the concentration of MgCl2.

[0060] 20μl system, the ratio is as follows:

[0061]

[0062] The PCR program is as follows:

[0063]

[0064] The result is as image 3 As shown, in MgCl 2 The amplification efficiency of the K-Ras gene is the highest under the condition of 2mM (the smaller the Cp value, the better the amplification efficiency), and this concentration is subsequently selected for the high-resolution melting curve (HRM) analysis of the 96-well plate.

[0065] (3) PCR comparison of the sealing effect of two LNA-Blockers

[0066] Using Primer 12 primers and Promega's Go-Taq TM The PCR reagent uses the K-Ras wild-type con...

Embodiment 3

[0080] Example 3 HRM Analysis Verification K-RAS Gene Mutation

[0081] ①Use Primer 12 primers and "LightCycler 480 high resolution melting master" to analyze common HRM on LightCycler 480 (Roche) machine and use LNA-blocker2 combined with HRM to analyze K-Ras gene point mutations.

[0082] Conventional HRM analysis PCR system and procedures are the same as above.

[0083] The template is: mutant and wild-type control plasmids are mixed in ratios of 1 / 0, 100 / 1, 10 / 1, 1 / 1, 1 / 10, 1 / 10, 1 / 100, and 0 / 1.

[0084] The HRM analysis procedure is as follows:

[0085] 95℃1min

[0086] 50℃1min

[0087] 72℃5seconds

[0088] continuous acquisition to 95℃at 30acquisitions per 1℃.

[0089] ②Use Primer 12 primers and "LightCycler 480 high resolution melting master" to analyze conventional HRM on LightCycler 480 (Roche) machine and use LNA-labeled probes combined with HRM to analyze K-Ras gene point mutations.

[0090] The HRM analysis PCR system and procedure combined with LNA-labeled p...

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Abstract

The invention discloses a K-Ras gene mutation detection method and applications thereof. According to the K-Ras gene mutation detection method, a locked nucleic acid labeled probe is adopted to detectK-Ras gene mutation. The probe is taken as a basis of the K-Ras gene mutation detection method, and combined fluorogenic quantitative PCR and high resolution solubility curve analysis are adopted. The K-Ras gene mutation detection method can be used for specific detection of the ratio of K-Ras gene mutation in DNA, and can be used for detection of novel K-Ras gene mutant sites.

Description

technical field [0001] The invention relates to a K-Ras gene point mutation detection method, comprising primers for amplifying K-Ras gene fragments, a locked nucleic acid labeling probe for detection, and the method in detecting the K-Ras gene mutation ratio in DNA application. Background technique [0002] The occurrence and development of malignant tumors is often a multi-factor and multi-step evolution process, involving the abnormal activation of multiple oncogenes and the inactivation of tumor suppressor genes. Among them, as the first oncogene isolated from human tumor cells, Ras gene plays an extremely important role in the occurrence of malignant tumors (Karnoub and Weinberg, Rasoncogenes: split personalities, 2008, 10.1038 / nrm2438). Orderly regulation of Ras protein activity is key to maintaining contact inhibition in normal cells. In normal cells, Ras will be activated only after growth factors bind to related receptors; while in the process of transformation fr...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/156
Inventor 金洪传冯利锋王娴林钰欣许文侠
Owner ZHEJIANG UNIV
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