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Method of detecting JAK2V617F mutation and its special primer and TaqMan MGB probe

A JAK2V617F and probe technology is applied in the field of detecting JAK2V617F mutation and its special primers and TaqMan-MGB probes, which can solve the problems of lack of primers and specific fluorescent probes.

Inactive Publication Date: 2009-06-10
PEOPLES HOSPITAL PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, there is currently no real-time fluorescent quantitative PCR detection technology specifically for detecting the JAK2V617F mutation on DNA or cDNA, because there are no suitable primers and specific fluorescent probes

Method used

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  • Method of detecting JAK2V617F mutation and its special primer and TaqMan MGB probe
  • Method of detecting JAK2V617F mutation and its special primer and TaqMan MGB probe
  • Method of detecting JAK2V617F mutation and its special primer and TaqMan MGB probe

Examples

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Embodiment 1

[0049] Example 1. Design of primers and TaqMan-MGB probes for detection of myeloproliferative disease JAK2V617F mutation

[0050] 1. Primers and TaqMan-MGB probes designed for the detection of myeloproliferative disease JAK2V617F mutation based on the genome gene of JAK2

[0051] According to the gene sequence (GenBank number of the wild type JAK2 gene: NT_008413, JAK2V617F mutant gene is the codon GTC of valine at position 617 of the wild-type gene mutated to the codon TTC of phenylalanine) and its reverse complementary sequence is designed with PrimerExpress Software 2.0 software. PCR primers, and two TaqMan-MGB probes were designed according to the reverse complementary sequences of the wild-type JAK2 kinase gene and the JAK2 kinase JAK2V617F mutant gene, and the 5' ends of the two TaqMan-MGB probes were respectively labeled with the reporter fluorescent group FAM( Red fluorescence) and VIC (green fluorescence), the 3' end is labeled with a non-luminescent quencher group, ...

Embodiment 2

[0062] Example 2. Detection of JAK2V617F mutation in myeloproliferative disease

[0063] 1. Detection of JAK2V617F mutation in myeloproliferative diseases using primers designed according to the JAK2 genome gene and TaqMan-MGB probe

[0064] Use the primers and TaqMan-MGB probes designed according to the JAK2 genome gene in step one of Example 1 for MPDs patients, including PV, ET, MF, unclassified MPDs, and AML, CML, ALL, CLL and non-Hodgkin Myeloproliferative disorders JAK2V617F For the detection of mutations, the normal donor bone marrow (NDM) was used as a control at the same time. The specific method is as follows:

[0065] 1) Extraction of genomic DNA from the sample to be tested

[0066] Genomic DNA was extracted from bone marrow samples of MPDs patients and normal bone marrow transplantation donors (see Table 3) under aseptic conditions according to the DNA zol kit (purchased from Gibco, USA) and referring to the kit instructions. The method was: anticoagulated with ...

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Abstract

The present invention discloses method of detecting JAK2V617F mutation and its special primer and TaqMan-MGB probe. The primer for detecting the genome DNA JAK2V617F mutation of myeloproliferative diseases is the primer pair shown in the SEQ ID No. 1 and SEQ ID No. 2 in the sequence list. The primer for detecting cDNA JAK2V617F mutation of myeloproliferative diseases is the primer pair shown in the SEQ ID No. 3 and SEQ ID No. 4 in the sequence list. The TaqMan-MGB probe has the DNA sequence shown in the SEQ ID No. 5 and SEQ ID No. 6 in the sequence list; and the DNA sequence has reporter fluorophore of different colors in the 5' end, and quenching group marked in the 3' end and connected dihydro clyclic indol porphyrin-tripeptide. The present invention provides one fast, reliable and accurate way for the diagnosis of MPDs, and can further provide the determined ratio between JAK2 mutation gene and wild JAK2 gene, so as to provide basis for treating observation.

Description

technical field [0001] The present invention relates to primers and probes for qualitative and quantitative detection of mutated bases by means of molecular biology methods, in particular to real-time fluorescent quantitative PCR (real-time fluorescent quantification PCR, RQPCR) technology for the detection of myeloproliferative disease JAK2V617F mutations Primers and TaqMan-MGB probes for qualitative and quantitative detection. Background technique [0002] Myeloproliferative disorders (MPDs) are a group of diseases caused by abnormal proliferation of one or more hematopoietic cell lines or connective tissue components. Research has shown that MPDs are strongly associated with blood disorders in which blood cells are overproduced or lose function. The four main MPDs are chronic myelogenous leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (idiopathic myelofibrosis, IMF). Among them, 95% of CML patients have BCR / ABL fusion ge...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 阮国瑞刘艳荣陈珊珊黄晓军李玲娣秦亚溱李金兰王峰蓉
Owner PEOPLES HOSPITAL PEKING UNIV
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