Primer combination for detecting human EGFR, KRAS and BRAF gene mutation and kit thereof

A kit and human detection technology, which is applied in DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problem that the site involved in the mutation is not the same site, cannot detect new mutations, and is not the same gene and other issues, to achieve the effect of optimizing primer design, reducing non-specific interference, and ensuring consistency

Inactive Publication Date: 2016-05-11
银丰基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the use of fluorescent probe technology can improve the specificity of detection, the cost of use is too high, and new mutations cannot be detected
In addition, the fluorescent probe is used to judge the Ct value of the fluorescence peak of the mutant group probe. Its defect is that there

Method used

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  • Primer combination for detecting human EGFR, KRAS and BRAF gene mutation and kit thereof
  • Primer combination for detecting human EGFR, KRAS and BRAF gene mutation and kit thereof
  • Primer combination for detecting human EGFR, KRAS and BRAF gene mutation and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0061] Example 1: Design and screening of ARMS primers for detecting mutations of human EGFR, KRAS and BRAF genes

[0062] When designing ARMS primers in the present invention, Taq enzyme lacks 3'-5' exonuclease activity. When the 3'end of the primer cannot be completely matched with the template, PCR amplification cannot be performed. The 3'end of the primer is designed to match the mutant template only, but not the normal template. The primer can only amplify the mutant template, but not the normal template, so as to achieve the purpose of typing.

[0063] The present invention also designs primer sequences for amplifying normal gene sites corresponding to mutation sites of human EGFR, KRAS, and BRAF genes, and uses the normal gene sites amplified by them as the second internal reference.

[0064] In this example, ARMS primers with mutations in human EGFR, KRAS, and BRAF genes were optimized and screened. The sequences are as follows:

[0065] EGFRExon-18-F-WT-ARMS: 5'-CTGAATTCAAAA...

Example Embodiment

[0100] Example 2: Investigation of the specificity of ARMS primers for detecting mutations of human EGFR, KRAS and BRAF genes

[0101] The positive samples containing EGFR, KRAS, and BRAF gene mutations were used as the detection objects, and the ARMS primers screened in Example 1 were used for detection.

[0102] The reaction system tested is: TaqHSDNA polymerase (1unit);

[0103] TaqHS DNA polymerase 10× buffer;

[0104] SYBR-Green-I(1:30000);

[0105] ARMS primer (0.1-0.5uM);

[0106] Template DNA (1-10ng);

[0107] 4 kinds of dNTP mixture (0.2mM);

[0108] High purity water.

[0109] PCR reaction conditions:

[0110] Select the SYBR fluorescence channel mode, and the fluorescence quantitative PCR reaction program is as follows:

[0111]

[0112] Note: *, fluorescence is collected in this step.

[0113] The result is that ARMS primers used to detect EGFR gene mutations can only specifically amplify positive samples containing EGFR gene mutations, but there is no amplification curve for posi...

Example Embodiment

[0123] Example 3: Sanger sequencing primers used to amplify EGFR, KRAS, and BRAF genes

[0124] The invention also provides Sanger sequencing primers for amplifying EGFR, KRAS, and BRAF genes, including amplification primers and sequencing primers.

[0125] Among them, the pair of amplification primers used to amplify the region of exon 18 of the EGFR gene is:

[0126] EGFRExon-18-F: 5'-CCGCTCGAGCTGAGGTGACCCTTGTCTC-3' (SEQ ID NO. 34);

[0127] EGFRExon-18-R: 5'-CGGGATCCCAGACCATGAGAGGCCCTG-3' (SEQ ID NO. 35);

[0128] The pair of amplification primers used to amplify the region of exon 19 of the EGFR gene is:

[0129] EGFRExon-19-F: 5'-GCCAGTTAACGTCTTCCTTCTC-3' (SEQ ID NO. 36);

[0130] EGFRExon-19-R: 5'-GATGTGGAGATGAGCAGGGTC-3' (SEQ ID NO. 37);

[0131] The pair of amplification primers used to amplify the region of exon 20 of the EGFR gene is:

[0132] EGFRExon-20-F: 5'-AAGCCACACTGACGTGCC-3' (SEQ IDNO.38);

[0133] EGFRExon-20-R: 5'-CCTGATTACCTTTGCGATCTGC-3' (SEQ IDNO.39);

[0134] The pair ...

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Abstract

The invention discloses an ARMS (Amplification Refractory Mutation System) primer for detecting human EGFR, KRAS and BRAF gene mutation. The ARMS primer is characterized in that sequences of the ARMS primer are as shown as in SEQ ID NO.1 to SEQ ID NO.31. The invention also discloses a kit containing the ARMS primer. According to the ARMS primer disclosed by the invention, a double-internal reference system is guided in an ARMS real-time fluorescence quantification technology, a normal site reference which is consistent with a mutation site is added for contrast expect a common internal reference, the defects of the prior art are avoided by the double-internal reference system and a corresponding double interpretation rule, the detecting accuracy is increased, and the error rate is reduced; a reagent which is used for carrying out Sanger sequencing on a sample is also contained in the detecting kit; by combining a Sanger sequencing technology with the ARMS real-time fluorescence quantification technology, a mutation site of a known gene can be detected, a new mutation site can also be found, and the detecting sensitivity is ensured.

Description

technical field [0001] The invention relates to a primer for detecting human EGFR, KRAS and BRAF gene mutations and a kit thereof, belonging to the technical field of molecular biology detection. Background technique [0002] According to the recommendation of NCCN, an authoritative organization in the field of cancer research and treatment in the world, lung cancer patients, especially non-small cell lung cancer (NSCLC) patients, are recommended to undergo EGFR gene mutation testing. The presence of activating mutations in EGFR has an important role in the selection of anticancer drugs for treatment. Studies have shown that some mutations of EGFR, especially its exon 19 deletion mutation, exon 21 point mutation (L858R), exon 18 point mutation, exon 20 point mutation (T790M), and The efficacy of tyrosine kinase inhibitors (TKIs) is highly correlated. In addition, in some lung cancer patients, mutations of EGFR gene and KRAS gene exist at the same time, and mutations in exo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156
Inventor 黄理刘海云李保伟
Owner 银丰基因科技有限公司
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