Primer and kit for detecting BRAF gene V600E mutation sites, and PCR method of kit

A mutation site and kit technology, applied in the fields of biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of the inability to detect clinical specimens on a large scale at the same time, the complex interpretation of kit results, and the high price of detection instruments. problems, to achieve the effect of avoiding site mismatch, fast detection speed, and high detection sensitivity

Active Publication Date: 2015-08-19
沈阳优吉诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] In view of this, the present invention provides a primer for detecting the V600E mutation site of the BRAF gene, a kit and a PCR method thereof, to at least solve the problems of complicated interpretation of results, high price of detection instruments, difficult operation, and certain false positives existing in previous kits. One or more problems such as negative and false positive, high testing cost, low clinical popularity, inability to test clinical specimens on a large scale at the same time

Method used

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  • Primer and kit for detecting BRAF gene V600E mutation sites, and PCR method of kit
  • Primer and kit for detecting BRAF gene V600E mutation sites, and PCR method of kit
  • Primer and kit for detecting BRAF gene V600E mutation sites, and PCR method of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Example 1: Preparation of wild type and mutant positive plasmids for BRAF gene V600E mutation site

[0120] First, we retrieved the gene sequence before and after the BRAF gene V600E mutation site from the gene bank, marked the mutation site with a double underline, and designed a pair of clones at appropriate positions upstream and downstream of the mutation site (marked with bold and underline) Primers, the amplified fragment is 205bp, including the V600E mutation site, and the gene sequence is shown as follows:

[0121] SEQ No.1:

[0122] gagaatatctgggcctacattgctaaaatctaatgggaaagttttaggttctcctataaacttaggaaagcatctcacctcatcctaacacatttcaagccccaaaaatcttaaaagcaggttatataggctaaatagaactaatcattgttttagacatacttattgactctaagaggaaagatgaagtactatgttttaaagaatattatattacagaattatagaaattagatctcttacctaaactcttcataatgcttgctctgataggaaaatgagatctactgtttt cctttacttactacacctcag atatatttcttcatgaagacctcacagtaaaaataggtgattttggtctagctacag t gaaatctcgatggagtgggtcccatcagtttgaacagttgtctggatccattttgt...

Embodiment 2

[0138] Example 2: Design and specificity screening of allele-specific primers (ASP)

[0139] For the BRAF-V600E mutation site as an example, design wild-type and a series of mutation-specific primers as follows:

[0140] BRAF-V600E-WT-F: aaaataggtgattttggtctagctactgt (SEQ No. 8)

[0141] BRAF-V600E-mut-F: aaataggtgattttggtctagctactga (SEQ No. 9)

[0142] BRAF-V600E-mut-F1: aataggtgattttggtctagctactga (SEQ No. 10)

[0143] BRAF-V600E-mut-F2: ataggtgattttggtctagctactga (SEQ No. 11)

[0144] BRAF-V600E-mut-F3: aataggtgattttggtctagctacaca (SEQ No. 12)

[0145] BRAF-V600E-mut-F4: ataggtgattttggtctagctacaca (SEQ No. 13)

[0146] BRAF-V600E-mut-F5: taggtgattttggtctagctacaca (SEQ No. 14)

[0147] Simultaneously design and synthesize Taqman-specific probe BRAF-V600E-Probe (SEQ No.15):

[0148] FAM-ctcgatggagtgggtcccatcagt-BHQ1, related primers and probes were synthesized in Sangon Bioengineering (Shanghai) Co., Ltd.

[0149] Then use the above 7 primers and BRAF-V600E-R (SEQ No....

Embodiment 3

[0152] Embodiment 3: ASP sensitivity screening

[0153] We then paired the No. 7 primer with the BRAF-V600E-R primer respectively, and used the mutant recombinant plasmid according to 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10, 0 for serial dilution, plus Taqman-specific probes, sensitivity verification was performed on a fluorescent quantitative PCR instrument. Mutant-specific primer No. 7 can detect 100 copies of the mutant, so this primer is the best primer for detecting the BRAF-V600E mutation site screened according to our method, as shown in Table 3.

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Abstract

The invention discloses a primer for detecting BRAF gene V600E mutation sites, and a PCR method of the kit. The primer comprises a wild type specific forward primer, a mutant type specific forward primer, and a reverse primer shared by the wild type specific forward primer and the mutant type specific forward primer, wherein the wild type specific forward primer has a sequence shown as SEQ No.17; the mutant type specific forward primer has a sequence shown as SEQ No.14; and the shared reverse primer has a sequence shown as SEQ No.16. The kit has the advantages of simple detection, rapidness, accuracy, low price and the like and provides a powerful tool for scientific research and clinical detection of BRAF gene V600E mutation sites and gene mutation analysis.

Description

technical field [0001] The invention relates to the field of molecular biology gene detection, and in particular provides primers, a kit and a PCR method for detecting the V600E mutation site of the BRAF gene, which are used for rapid detection of the V600E mutation site of the BRAF gene. Background technique [0002] The BRAF gene, located on chromosome 7q34, encodes a serine / threonine protein kinase and is a member of the RAF family. It was first discovered and cloned by Ikawa et al. Homology, so called BRAF. The BRAF protein consists of 783 amino acids, and there are three conserved regions, CR1, CR2 and CR3, from the N-terminus to the C-terminus. Among them, the CR1 region consists of a RAS protein binding region and a cysteine-rich region, both of which can bind to RAS; CR2 is rich in serine / threonine, which regulates phosphorylated RAF kinase activity; CR3 region is ATP-binding The site and activation region contain tyrosine and serine residues and have multiple phos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/106C12Q2600/156
Inventor 高劲松张英杰魏欣芳李星颐魏潇魏奇
Owner 沈阳优吉诺生物科技有限公司
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