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Method and kit for detecting the 12th and 13th codon mutation sites of kras gene

A technology of mutation sites and codons, which is applied in the kit for detecting mutation sites of the 12th and 13th codons of the Kras gene, and in the field of mutation sites of the 13th codons, which can solve the cumbersome operation of next-generation sequencing, limited detection capabilities, and analytical problems. Complicated problems, to achieve the effect of low cost, short time-consuming, simple and fast operation

Active Publication Date: 2020-06-05
上海伯豪生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method is a fluorescent PCR detection method based on the ARMS-qPCR system with optimized amplification reaction primers, fluorescent probes and blocking probes (block probes), which can overcome the low throughput, time-consuming, and detection capabilities of the existing generation sequencing technology. Limited problems can also overcome the shortcomings of next-generation sequencing, such as cumbersome operations, complex analysis, and high cost

Method used

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  • Method and kit for detecting the 12th and 13th codon mutation sites of kras gene
  • Method and kit for detecting the 12th and 13th codon mutation sites of kras gene
  • Method and kit for detecting the 12th and 13th codon mutation sites of kras gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Using the plasmid template containing the 12th and 13th codon mutation sites of the Kras gene (corresponding to each site to be tested), using the primers and probes in Table 2, qPCR was performed to optimize and select the best ARMS primers.

[0057] Wherein, the wild-type plasmids and mutant-type plasmids involved in the following tables can be prepared according to conventional plasmid construction methods and PCR cloning methods.

[0058] 1) Plasmid treatment and extraction:

[0059] Plasmid extraction was carried out using the plasmid extraction kit of TIANGEN (HighPure Plasmid Kit, DP116). For details, please refer to the product manual. The extracted DNA was dissolved in Tris-HCl (10mmol / L, pH8.0), and the quality of the sample was detected by an ultraviolet spectrophotometer and the concentration was determined. Then, dilute the sample to 2000copies / uL. Take 5 μL for PCR reaction.

[0060] 2) Perform PCR amplification according to the following amplification ...

Embodiment 2

[0086] Using the present invention to detect DNA samples of mutant cell lines, culture the corresponding cell line cells containing each mutation site, extract DNA, use the specific ARMS primers and fluorescent probe PCR system of the present invention to detect it, and simultaneously perform the minimum detection limit determination.

[0087] Proceed as follows:

[0088] 1) Sample processing and DNA extraction:

[0089] Use a commercial DNA extraction kit to extract sample DNA, and refer to the kit instructions for specific operations.

[0090] Samples were diluted to 10ng / μL.

[0091] 2) Perform PCR amplification according to the following amplification system (total volume 40 μL)

[0092]

[0093] Wherein, the primers in the above PCR amplification are the primers shown in SEQ ID NO.1-9, and the probe is the probe shown in SEQ ID NO.17.

[0094] In addition, 2×Premix Ex Taq (Probe qPCR) in PCR amplification was from TAKARA Company, and water was from LIFETECH Company....

Embodiment 3

[0104] Using the present invention to detect tissue slice samples, 66 tissue slice samples were extracted for DNA, then detected using the specific ARMS primers and fluorescent probe PCR system of the present invention, and verified by first-generation sequencing. Using the present invention to detect tissue slice samples, after extracting DNA from 79 tissue slice samples, use the specific ARMS primers and fluorescent probe PCR system of the present invention to detect them, and carry out next-generation sequencing verification.

[0105] Proceed as follows:

[0106] 1) Sample processing and DNA extraction:

[0107] Use a commercial DNA extraction kit to extract sample DNA, and refer to the kit instructions for specific operations.

[0108] Samples were diluted to 10ng / μL.

[0109] 2) Perform PCR amplification according to the following amplification system (total volume 40 μL)

[0110]

[0111] Wherein, the primers in the above-mentioned PCR amplification are the primers...

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Abstract

The invention discloses a method for detecting mutation sites of codons 12 and 13 of a Kras gene and a kit of the method. The method comprises the following steps: (1) extracting DNA of a sample as a DNA template; (2) carrying out fluorescence PCR amplification on the DNA template by virtue of a group of ARMS primers, a fluorescence probe and a closed probe, wherein upstream primers in the ARMS primers are selected from any one of sequences represented by SEQ ID NO.1-8, downstream primers in the ARMS primers are selected from a sequence represented by SEQ ID NO.9, the fluorescence probe is represented by SEQ ID NO.17, and the closed probe is represented by SEQ ID NO.18; and (3) detecting the fluorescence intensity of a reaction system in the fluorescence PCR amplification, and judging the mutation of the mutation sites of the codons 12 and 13 of the Kras gene according to a cycle index CT value required when the fluorescence intensity reaches a set threshold. The kit comprises the ARMS primers, the fluorescence probe and the closed probe. According to the method and the kit, the disadvantages of low flux, long consumed time, limited detection capacity and like of an existing sequencing technique can be overcome; and the method and the kit have the advantages of rapidness, high flux and sensitivity, good specificity and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for detecting mutation sites of the 12th and 13th codons of Kras gene. This kit is used for the qualitative detection of 7 common mutations on codon 12 and codon 13 of human KRAS gene in paraffin-embedded pathological tissue samples. In addition, the invention also relates to a method for detecting mutation sites of the 12th and 13th codons of the Kras gene. Background technique [0002] KRAS is a proto-oncogene, about 35kb in length, located on chromosome 12. It is a member of the RAS gene family. The protein encoded by this gene is related to the formation, proliferation, migration, spread and angiogenesis of tumors. The KRAS gene has a high probability of mutation in various tumors. Common mutation sites are codons 12 and 13 of exon 2 of the KRAS gene. The seven most common mutation hotspots are: G12C, G12S, G12R, and G12V , G12D, G12A, G13D, these seven mutati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12Q1/6869
CPCC12Q1/686C12Q1/6869C12Q1/6886C12Q2600/156C12Q2535/137C12Q2563/107C12Q2545/114C12Q2535/122
Inventor 徐晓晶赵莹姚琴琴陆凌佳贺华
Owner 上海伯豪生物技术有限公司