Primer, kit as well as PCR method for detecting of D816V mutation site of C-KIT gene

A D816V, mutation site technology, applied in the field of molecular biology gene detection, can solve the problems of inability to simultaneously detect large-scale clinical specimens, complicated interpretation of kit results, and low clinical popularity, so as to avoid site mismatch and detection. The effect of fast speed and good 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

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Problems solved by technology

[0020] In view of this, the present invention provides a primer for detecting the D816V mutation site of the C-KIT gene, a kit and a PCR method thereof, to at least solve the complex interpretation of results, high price of detection instruments, difficult operation, and the existence of existing kits in the past. Certain false negatives and false positives, high cost of testing, low clinical popularity, inability to test clinical specimens on a large scale at the same time, etc. One or more problems

Method used

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  • Primer, kit as well as PCR method for detecting of D816V mutation site of C-KIT gene
  • Primer, kit as well as PCR method for detecting of D816V mutation site of C-KIT gene
  • Primer, kit as well as PCR method for detecting of D816V mutation site of C-KIT gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1: Preparation of wild-type and mutant-type positive plasmids for the C-KIT gene D816V mutation site

[0122] C-KIT encodes KIT receptors and is involved in regulating gene expression, controlling cell growth and proliferation. Mutations in C-KIT gene can lead to malignant tumors such as acute myeloid leukemia, gastrointestinal stromal tumor and testicular cancer. Clinical studies have shown that the mutation of the C-KIT gene in gastrointestinal stromal tumors is closely related to the efficacy of Imatinib molecular targeted therapy. Patients with C-KIT exon 11 mutation had the best curative effect, followed by patients with exon 9 mutation, and GIST patients without gene mutation had the worst curative effect. The American Cancer Comprehensive Network (NCCN) "Guidelines for the Clinical Treatment of Soft Tissue Sarcoma" proposes that before receiving targeted drug therapy, GIST patients should be tested for C-KIT gene mutations, and whether to use targeted dru...

Embodiment 2

[0140] Example 2: Design and specificity screening of allele-specific primers (ASP)

[0141] For the C-KIT gene D816V mutation site, wild-type and a series of mutation-specific primers were designed as follows:

[0142] C-KIT-D816V-WT-F: gatttgtgattttggtctagccagtga (SEQ No. 8)

[0143] C-KIT-D816V-mut-F: gatttgtgattttggtctagccagtgt (SEQ No. 9)

[0144] C-KIT-D816V-mut-F1: atttgtgattttggtctagccagtgt (SEQ No. 10)

[0145] C-KIT-D816V-mut-F2: tttgtgattttggtctagccagtgt (SEQ No. 11)

[0146] C-KIT-D816V-mut-F3: gatttgtgattttggtctagccagact (SEQ No. 12)

[0147] C-KIT-D816V-mut-F4: atttgtgattttggtctagccagact (SEQ No. 13)

[0148] C-KIT-D816V-mut-F5: tttgtgattttggtctagccagact (SEQ No. 14)

[0149] Simultaneously design and synthesize Taqman-specific probe C-KIT-D816V-Probe:

[0150] SEQ No. 15: FAM-aaaggaaacgtgagtacccattctctgctt-BHQ1.

[0151] Relevant primers and probes were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

[0152] Then use the above 7 primers to pai...

Embodiment 3

[0154] Embodiment 3: ASP sensitivity screening

[0155] Then use the mutant primers after screening to pair with the common downstream primer SEQ No.16 of the C-KIT gene D816V mutation site, and use 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. The mutation-specific primer can detect 100 copies of the mutant, so this primer is the best primer for detecting the D816V mutation site of the C-KIT gene screened according to our method, as shown in Table 3.

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Abstract

The invention discloses a primer, a kit as well as a PCR method for detecting a D816V mutation site of a C-KIT gene. The primer comprises a wild-type specific forward primer, a mutant-type specific forward primer and a reverse primer which are 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 in SEQ No.17, the-mutant type specific forward primer has a sequence shown in SEQ No.14, and the shared reverse primer has a sequence shown in SEQ No.16. The kit has the advantages of being simple, rapid, accurate and cheap in detection; and a powerful instrument is provided for scientific research and clinical D816V site typing and gene mutation analysis of the C-KIT gene.

Description

technical field [0001] The invention relates to the field of molecular biological gene detection, and in particular provides a primer for detecting C-KIT gene mutation, a kit and a PCR method thereof, which are used for rapid detection of the D816V mutation site of the C-KIT gene. Background technique [0002] The C-KIT gene is a proto-oncogene, and the encoded KIT receptor is a type III tyrosine kinase. After the receptor binds to the ligand-stem cell factor (SCF), it activates downstream signals by forming a dimer, including Ras / Raf / MAPK pathway, Akt / PI3K pathway, etc., and finally activates transcription factors in the cytoplasm to regulate genes Expression, control of cell growth and proliferation. [0003] Under normal circumstances, KIT receptors are activated with the participation of stem cell factors. If the C-KIT gene is mutated, the activation of the KIT receptor does not require the participation of the ligand SCF, which will cause the continuous proliferation ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/156
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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