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Primers, kit and pcr method for detecting jak2 gene v617f polymorphism

A V617F, locus polymorphism technology, applied in the field of molecular biology gene detection, can solve the problems of inability to detect large-scale clinical specimens at the same time, complicated interpretation of kit results, and low clinical popularity, so as to avoid locus mismatch. , the detection speed is fast, the effect of good sensitivity

Active Publication Date: 2018-06-26
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

[0015] In view of this, the present invention provides a primer for detecting the V617F polymorphism site of the JAK2 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 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 jak2 gene v617f polymorphism
  • Primers, kit and pcr method for detecting jak2 gene v617f polymorphism
  • Primers, kit and pcr method for detecting jak2 gene v617f polymorphism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1: Preparation of wild type and mutant positive plasmids for JAK2 gene V617F polymorphic site

[0119] JAK is a non-receptor tyrosine protein kinase, among which JAK2 mutation is closely related to myeloproliferative diseases. JAK2 gene V617F mutation can lead to abnormal activation of JAK-STAT signaling pathway, resulting in abnormal proliferation of bone marrow cells. In the 2008 World Health Organization (WHO) classification system, JAK2 mutation has become the main diagnostic index of chronic myeloproliferative disease (MPD).

[0120] First, we called out the gene sequence before and after the V617F polymorphic site of the JAK2 gene from the gene bank, and marked the polymorphic site with a double underline, at the appropriate position upstream and downstream of the V617F polymorphic site of the JAK2 gene ( Marked in bold and underlined), design a pair of cloning primers, the amplified fragment is 228bp, including the mutation site, the gene sequence is show...

Embodiment 2

[0137] Example 2: Design and specificity screening of allele-specific primers (ASP)

[0138] For JAK2-V617F, design wild-type and a series of mutant-specific primers as follows:

[0139] JAK2-V617F-WT-R: ttacttactctcgtctccacacac (SEQ No. 8)

[0140] JAK2-V617F-mut-R: tttacttactctcgtctccacacaa (SEQ No. 9)

[0141] JAK2-V617F-mut-R1: tacttactctcgtctccacacaa (SEQ No. 10)

[0142] JAK2-V617F-mut-R2: acttactctcgtctccacacaa (SEQ No. 11)

[0143] JAK2-V617F-mut-R3: ctttacttactctcgtctccacagga (SEQ No. 12)

[0144] JAK2-V617F-mut-R4:cttacttactctcgtctccacagga (SEQ No. 13)

[0145] JAK2-V617F-mut-R5: ctacttactctcgtctccacagga (SEQ No. 14)

[0146] Simultaneously design and synthesize Taqman-specific probes:

[0147] SEQ No. 15: FAM-tgaagcagcaagtatgatgagcaagc-BHQ1.

[0148] Relevant primers and probes were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

[0149] Then use the above 7 primers to pair with the common downstream primer JAK2-V617F-F: 5'-ggacaacagtcaaacaacaattc-3...

Embodiment 3

[0151] Embodiment 3: ASP sensitivity screening

[0152] Then use No. 7 mutant primers to pair with the common downstream primer SEQ No.16 of the JAK2 gene, and use mutant recombinant plasmids 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 No. 7 mutation-specific primer can detect 100 copies of the mutant, so this primer is the best primer for detecting the V617F polymorphism site of the JAK2 gene screened according to our method, as shown in Table 3.

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Abstract

The invention discloses primers and a kit for detecting JAK2 gene V617F site polymorphism, and a PCR (polymerase chain reaction) method thereof. The primers comprise a wild specific forward primer, a mutant specific forward primer and a common reverse primer for the wild specific forward primer and mutant specific forward primer, wherein the wild specific forward primer is disclosed as SEQ NO.17, the mutant specific forward primer is disclosed as SEQ NO.14, and the common reverse primer is disclosed as SEQ NO.16. The kit has the advantages of simple detection, high speed, high accuracy, low price and the like, and provides a powerful tool for scientific research and analysis on clinical JAK2 gene V617F site typing and gene mutation.

Description

technical field [0001] The invention relates to the field of molecular biological gene detection, and in particular provides a primer for detecting JAK2 gene polymorphism, a kit and a PCR method thereof, which are used for rapid detection of the V617F polymorphism site of the JAK2 gene. Background technique [0002] JAK is a non-receptor tyrosine protein kinase. So far, four family members have been found, namely JAK1, JAK2, JAK3 and JAK4. STAT is the direct substrate of JAK, which can directly transmit the signal to the nucleus and regulate the expression of specific genes. The JAK-STAT signaling pathway is a signaling pathway closely related to cell growth, proliferation, and differentiation, and also participates in signal transduction in hematopoietic and immune systems, and the kinase JAK plays a key role in the activation of the entire signaling pathway. So far, many JAK gene point mutations have been found in human leukemia cells, some of which can lead to the contin...

Claims

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Application Information

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