Prime group, probe group and kit for detecting Kras gene mutation

A detection kit and the technology of the kit are applied in the fields of biochemical equipment and methods, recombinant DNA technology, and the determination/inspection of microorganisms, which can solve the problems of expensive instruments, low popularization and utilization, and achieve objective interpretation of results and flexible application. , the effect of high detection sensitivity

Active Publication Date: 2016-07-27
CREATIVE BIOSCIENCES (GUANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method also has defects such as expensive instruments and low popularity and utilization.

Method used

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  • Prime group, probe group and kit for detecting Kras gene mutation
  • Prime group, probe group and kit for detecting Kras gene mutation
  • Prime group, probe group and kit for detecting Kras gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] The design of embodiment 1 primer and probe

[0072] 1. Primer design: The 7 common mutation types of Kras gene are shown in Table 1.

[0073] Table 1 Seven common mutation types of Kras gene

[0074] Mutation type marker in the present invention

mutation name

amino acid changes

base change

34T

12CYS

G12C

c.34G>T

34A

12SER

G12S

c.34G>A

34C

12ARG

G12R

c.34G>C

35T

12VAL

G12V

c.35G>T

35A

12ASP

G12D

c.35G>A

35C

12ALA

G12A

c.35G>C

38A

13ASP

G13D

c.38G>A

[0075] In the present invention, among the primers for detecting the above seven kinds of mutations, the same reverse primers are shared, and specific forward primers are used respectively.

[0076] (1) The common reverse primer is:

[0077] Kras-Rev-Rp: GGCCTGCTGAAAATGACTG

[0078] (2) A plurality of forward primers were respectively designed for 7 different mutat...

Embodiment 2

[0094] Further optimization of embodiment 2 primers and probes

[0095] 1. Forward primer optimization

[0096] (1) The primers were further optimized using the above PCR reaction conditions.

[0097] The PCR templates are the plasmid templates of the mutant type and the wild type respectively, and the specific preparation process is as follows: refer to the reference sequences of the wild type of the Kras gene and 7 common mutant types queried in the COSMIC database, and synthesize the wild type and the wild type in sequence according to the reference sequences. Eight kinds of artificial nucleic acid sequences containing mutation sites are connected to plasmid vectors, and after screening, plasmids loaded with wild-type and seven kinds of mutant sequences are obtained. The concentration of the plasmid template is on the order of 10,000 copies. The results are shown in Table 6.

[0098] Table 6 Primer optimization results

[0099]

[0100]

[0101]

[0102] Note: "-...

Embodiment 3

[0113] The assembly of embodiment 3Kras gene mutation detection kit

[0114] 1. According to the above research results, the present invention has successfully assembled a kit for detecting Kras gene mutation with high sensitivity and high accuracy, including the following components:

[0115] (1) Primers: Forward primers are shown in Table 8:

[0116] Table 8

[0117]

[0118] Reverse primer (SEQ ID NO.8): Kras-Rev-Rp: GGCCTGCTGAAAATGACTG.

[0119] (2) probe, as shown in table 9:

[0120] Table 9

[0121]

[0122]

[0123] (3) dNTPs required for the PCR reaction, preferably 10 mM dNTPs.

[0124] (4) Enzyme; preferably Taq hot start enzyme (DNA polymerase); its concentration is preferably 5 U / μl.

[0125] (5) Reaction buffer, preferably 5*enzyme buffer.

[0126] (6)Mg 2+ , preferably 25mM Mg 2+ .

[0127] (7) Nuclease-free water.

[0128] 2. The PCR reaction system and reaction procedures when using the above kit are shown in Table 4 and Table 5 in Example 1...

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Abstract

The invention discloses a prime group, probe group and kit for detecting Kras gene mutation. The prime group comprises forward primers, reverse primers and probes which are used for detecting 7 Kras gene mutation types, wherein the forward primers respectively are 34A-Rev-Fp10, 35A-Rev-Fp4, 38A-Rev-Fp13, 34C-Rev-Fp, 34T-Rev-Fp, 35C-Rev-Fp and 35T-Rev-Fp, the reverse primers are Kras-Rev-Rp, and the probes respectively are 34A-Rev-Pb4, 35A-Rev-Pb2, 38A-Rev-Pb3, 34C-Rev-Pb, 34T-Rev-Pb, 35C-Rev-Pb and 35T-Rev-Pb. The prime group, the probe group and the kit are high in detection sensitivity and accuracy of Kras gene mutation, good in specificity and capable of respectively and simultaneously detecting 7 different mutation types.

Description

technical field [0001] The invention belongs to the technical field of gene detection. More specifically, it relates to a set of primers and probes for detecting Kras gene mutation and a kit thereof. Background technique [0002] The RAS gene was first discovered in the early 1980s. It was a transformation gene isolated from a human bladder cancer cell line, which could cause malignant transformation of NIH3T3 cells (mouse embryonic fibroblast cell line), while normal human tissue The DNA extracted from it had no such effect. The RAS gene is quite conserved in evolution and widely exists in various eukaryotes such as mammals, Drosophila, fungi, nematodes and yeast, suggesting that it has important physiological functions. [0003] There are three genes in the RAS gene family associated with human tumors—Hras, ​​Kras, and Nras, which are located on chromosomes 11, 12, and 1, respectively. Kras is also known as the p21 gene because it encodes a 21kD RAS protein. Among the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 邹鸿志牛智通
Owner CREATIVE BIOSCIENCES (GUANGZHOU) CO LTD
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