Primers and kit for detecting JAK2 gene V617F site polymorphism, and PCR (polymerase chain reaction) method thereof

A technology of V617F and site polymorphism, which is applied in the field of molecular biology gene detection, can solve the problems of inability to detect clinical specimens on a large scale at the same time, the interpretation of kit results is complicated, and the price of detection instruments is high, so as to avoid site mismatch, The effect of fast detection speed and high detection sensitivity

Active Publication Date: 2015-08-05
沈阳优吉诺生物科技有限公司
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  • Abstract
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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, dif

Method used

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  • Primers and kit for detecting JAK2 gene V617F site polymorphism, and PCR (polymerase chain reaction) method thereof
  • Primers and kit for detecting JAK2 gene V617F site polymorphism, and PCR (polymerase chain reaction) method thereof
  • Primers and kit for detecting JAK2 gene V617F site polymorphism, and PCR (polymerase chain reaction) method thereof

Examples

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Example Embodiment

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

[0119] JAK is a non-receptor type tyrosine protein kinase. JAK2 mutation is closely related to myeloproliferative diseases. JAK2 gene V617F mutation can cause abnormal activation of JAK-STAT signaling pathway, leading to abnormal proliferation of bone marrow cells. In the 2008 World Health Organization (WHO) classification system, JAK2 mutation became the main diagnostic indicator of chronic myeloproliferative disease (MPD).

[0120] First, we call out the gene sequence before and after the V617F polymorphism site of JAK2 gene from the gene bank, and mark the polymorphism site with double underline, in the appropriate position upstream and downstream of the V617F polymorphism site of JAK2 gene ( (Marked in bold and underlined), design a pair of cloning primers, the amplified fragment is 228bp, the mutation site is included, the gene sequence is shown as follows, S...

Example Embodiment

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

[0138] For JAK2-V617F, wild-type and a series of mutation-specific primers are designed 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 Shenggong Bioengineering (Shanghai) Co., Ltd.

[0149] Then use the above 7 primers and the common downstream primer of JAK2 gene JAK2-V617F-F: 5'-ggacaacagtcaaacaacaattc-3' (SEQ No. 1...

Example Embodiment

[0151] Example 3: ASP Sensitivity Screening

[0152] Then use the No. 7 mutant primer to pair with the common downstream primer SEQ No. 16 of the JAK2 gene, and use the mutant recombinant plasmid according to 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10,0 as a gradient dilution, plus Taqman specific probe, the sensitivity verification on the fluorescence quantitative PCR instrument. The mutation-specific primer No. 7 can detect 100 copies of the mutant, so this primer is the best primer for detecting the V617F polymorphic site of JAK2 gene 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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IPC IPC(8): C12Q1/68C12N15/11
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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