Primers and probes for detecting mutation of cancer gene BRAFV600E
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An oncogene and probe technology, which is applied in the field of multiple primers and probes for the detection of oncogene BRAFV600E mutation, to achieve strong specificity, convenient quality control, and avoid false negative results
Inactive Publication Date: 2011-11-16
ZHEJIANG UNIV
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Commonly used BRAF mutation detection methods mainly include direct sequencing, restriction fragment length polymorphism (RFLP), and single-strand conformation polymorphism (SSCP), but all of them have certain limitations.
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[0035] (1) The mutation-specific upstream primer BRAFFE-1 sequence is:
[0036] 5'-GTGATTTTGGTCTAGCTACTGA-3'.
[0037] (2) The sequence of the mutant non-specific upstream primer BRAFFV-1 is:
[0038] 5-AGTAAAAATAGGTGATTTTGGTCTAG-3'.
[0039] (3) The general downstream primer BRAFR-1 sequence is:
[0053] (1) The mutation-specific upstream primer BRAFFE-1 sequence is:
[0054] 5'-AAAAATAGGTGATTTTGGTCTAGCTACATA-3'.
[0055] (2) The sequence of the mutant non-specific upstream primer BRAFFV-1 is:
[0056] 5'-AAAAATAGGTGATTTTGGTCTAGCTACA-3'.
[0057] (3) The general downstream primer BRAFR-1 sequence is:
[0058] 5'-TAGTTGAGACCTTCAATGACTTTCTTAGTAA-3'.
[0059] (4) TaqMan probe sequence:
[0060] 5'-FAM-ACAACTGTTCAAACTGATGGGACC-BHQ1-3'.
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Abstract
The invention relates to the field of biotechnology and aims to provide multiple primers and probes for detecting mutation of a cancer gene BRAFV600E. The primers and probes provided by the invention respectively comprise a mutation specific forward primer, a mutation non-specific forward primer, universal reverse primers and universal probes shown in SEQ NO.1-11. The detection sensitivity of themutation specific primer and probe provided by the invention can reach 500 copies/ml, thus the sensitivity is good. If a sample does not contain the cancer gene BRAFV600E, the ct value of the amplification curve of the sample is greater than or equal to 36, and the specificity is strong. Because the mutation non-specific primer is simultaneously designed in the invention to be used for detecting the total template quantity of samples, the false negative result is avoided and the quality control is convenient.
Description
technical field [0001] The invention belongs to the field of biological technology, in particular to a method for detecting cancer gene BRAF V600E Multiple primers and probes for mutations. Background technique [0002] The BRAF gene is an oncogene, which belongs to the RAF family with the ARAF and RAF1 (CRAF) genes. It is located at 7q34 and is about 190kb long. The BRAF protein translated by it consists of 783 amino acids and has three conserved regions, CR1, CR2 and CR3. BRAF protein is a member of the serine-threonine protein kinase raf / mil family and an important regulator in the upstream of the Ras-Raf-MEK-ERK signaling pathway. The BRAF protein is located at the entrance of the MAPK / ERK pathway, which connects receptors and RAS proteins on the cell surface to transcription factors in the nucleus through MEK and ERK, thereby regulating cell differentiation and division. When this pathway is abnormally activated, it can promote cell proliferation and growth, and even...
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