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
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[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...
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[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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