Primers, kit and pcr method for detecting d842v polymorphic site of pdgfra gene

A technology of polymorphic sites and kits, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of inability to detect clinical specimens on a large scale at the same time, low clinical popularity, and the price of testing instruments Advanced problems, to achieve the effect of avoiding site mismatch, fast detection speed and high sensitivity

Active Publication Date: 2018-04-27
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

[0017] In view of this, the present invention provides a primer for detecting the D842V polymorphism site of the PDGFRA gene, a kit and a PCR method thereof, to at least solve the complex interpretation of the results of the previous kits, the high price of detection instruments, the difficulty of operation, and the existence of Certain false negatives and false positives, high testing costs, 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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  • Primers, kit and pcr method for detecting d842v polymorphic site of pdgfra gene
  • Primers, kit and pcr method for detecting d842v polymorphic site of pdgfra gene
  • Primers, kit and pcr method for detecting d842v polymorphic site of pdgfra gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Example 1: Preparation of wild-type and mutant positive plasmids for PDGFRA gene D842V polymorphic site detection

[0121] First, we called out the gene sequence before and after the D842V polymorphic site of the PDGFRA gene from the gene bank, and marked the polymorphic site with a double underline, and placed it at the appropriate position upstream and downstream of the D842V polymorphic site of the PDGFRA gene (in bold word and underline), design a pair of cloning primers, the amplified fragment is 120bp, including the mutation site, the gene sequence is shown below, SEQ No.1:

[0122] agctacagatggcttgatcctgagtcatttcttccttttccatgcagtgtgt ccaccgtgatctggctgctc gcaacgtcctcctggcacaaggaaaaattgtgaagatctgtgactttggcctggccagag a catcatgcatgattc gaacta tgtgtcgaaaggcagtgt acgtcctcacttccctcactggtcaggctcatcctccttcactttaatctctaaagtcaggtgttgcttctagagattcggtgcctgttttttaaaacatcaatagattt

[0123] The experimental steps are as follows:

[0124] 1) Genomic DNA extraction

[012...

Embodiment 2

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

[0139] Aiming at the D842V polymorphic site of PDGFRA gene, wild-type and a series of mutation-specific primers were designed as follows:

[0140] PDGFRA-D842V-WT-R: cacatagttcgaatcatgcatgaagt (SEQ No. 8)

[0141] PDGFRA-D842V-mut-R: cacatagttcgaatcatgcatgaaga (SEQ No. 9)

[0142] PDGFRA-D842V-mut-R1: acatagttcgaatcatgcatgaaga (SEQ No. 10)

[0143] PDGFRA-D842V-mut-R2: catagttcgaatcatgcatgaaga (SEQ No. 11)

[0144] PDGFRA-D842V-mut-R3: cacatagttcgaatcatgcatgatca (SEQ No. 12)

[0145] PDGFRA-D842V-mut-R4: acatagttcgaatcatgcatgatca (SEQ No. 13)

[0146] PDGFRA-D842V-mut-R5: catagttcgaatcatgcatgatca (SEQ No. 14)

[0147] Simultaneously design and synthesize Taqman-specific probes:

[0148] SEQ No.15: FAM-ccaggccaaagtcacagatcttcacaat-BHQ1, related primers and probes were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

[0149] Then use the above 7 primers to pair with the PDGFRA-D8...

Embodiment 3

[0151] Embodiment 3: ASP sensitivity screening

[0152] Then use the mutant primers to pair with the common downstream primer SEQ No.16 of the PDGFRA gene D842V polymorphic 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. Mutation-specific primers can detect 100 copies of mutants, so this primer is the best primer for detecting the D842V polymorphism site of PDGFRA gene screened according to our method, as shown in Table 3.

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Abstract

The invention discloses a primer and a kit for detecting PDGFRA (platelet-derived growth factor receptor alpha) gene D842V polymorphic sites and a PCR (polymerase chain reaction) method of the primer and the kit. The primer comprises a wild specific sense primer, a mutant type specific sense primer, and a reverse primer shared by the wild specific sense primer and the mutant type specific sense primer. The wild specific sense primer has an SEQ No.17 sequence, the mutant type specific sense primer has an SEQ No.14 sequence, and the shared reverse primer has an SEQ No.16 sequence. The kit has the advantages of simplicity in detection, quickness, accuracy, low cost and the like, and powerful tools are provided for scientific research and clinic analysis of the PDGFRA gene D842V polymorphic sites.

Description

technical field [0001] The invention relates to the field of molecular biological gene detection, and in particular provides a primer for detecting the D842V polymorphic site of the PDGFRA gene, a kit and a PCR method thereof, which are used for rapid detection of the D842V polymorphic site of the PDGFRA gene. Background technique [0002] Platelet-derived growth factor (PDGF) is a pro-angiogenic factor, and PDGF is a potent mitogen of various mesenchymal cells such as fibroblasts, vascular smooth muscle cells, glomerular vascular cells, glial cells, endothelial cells, etc. Original and important vascular growth factor, produced by platelets, histiocytes and certain tumor cells. Platelet-derived growth factor receptor (PDGFR) is a member of the receptor tyrosine protein kinase family, which can promote cell chemotaxis, division and proliferation, and play an active and important role in physiological processes such as body growth and development, wound repair, etc. And clos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2561/101
Inventor 高劲松张英杰李星颐王箫笛魏潇魏奇
Owner 沈阳优吉诺生物科技有限公司
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