Primers and method for detecting MPL gene mutation

A gene and detection result technology, applied in the field of primers for detecting MPL gene mutations, can solve problems such as complex reaction systems, achieve high sensitivity, good specificity, and improve detection efficiency

Inactive Publication Date: 2015-12-16
GUANGZHOU KINGMED DIAGNOSTICS CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Chinese patent application 201210374903.7 discloses a kit for detecting the W515 site mutation of the MPL gene, which includes site mutation-specific primers, internal reference gene ABL primers and fluorescent probes, etc., for the detection of the W515 site mutation of the MPL gene There are disadvantages that the reaction system is complex and requires a variety of primers and fluorescent quantitative PCR instruments

Method used

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  • Primers and method for detecting MPL gene mutation
  • Primers and method for detecting MPL gene mutation
  • Primers and method for detecting MPL gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Primer

[0020] The inventors designed a large number of primers for exon 10 of the MPL gene, and selected primers with good specificity through optimization and comparison of primer reaction conditions.

[0021]

Embodiment 2

[0022] The specificity of embodiment two primers

[0023] The primers provided by the present invention were blasted in UCSC, and the amplified fragment of the exon 10 primer of the MPL gene was located between chr1:43814796-43815124, with a length of 329bp. No other homologous genes, the results are as follows figure 1 As shown, it is consistent with the reference sequence of MPL gene.

[0024] The primers in Table 1, the PCR amplification system in Table 2 and the PCR amplification conditions in Table 3 are used to amplify the detection sample, and the amplified product is analyzed by gel electrophoresis. The results are as follows figure 2 shown. The results showed that the specificity of the amplified product was high, and no non-specific amplified bands were produced.

Embodiment 3

[0025] Example 3 Detection of Exon 10 Mutation of MPL Gene

[0026] Extract DNA samples from EDTA-anticoagulated peripheral blood. The extraction method refers to the instructions of TIANampBloodDNAKit (purchased from Tiagen, product number: DP318). The DNA samples are diluted to 100ng / μL and set aside.

[0027] PCR amplification using Q5 ? Hot start ultra-fidelity 2XMasterMix (purchased from NEB Company, item number: M0494L), the PCR amplification system is shown in Table 2, and the PCR amplification conditions are shown in Table 3. The concentration ratio of the upstream primer of exon 10 and the downstream primer of exon 10 was 1:1, and the concentration of the upstream primer of exon 10 and the downstream primer of exon 10 were both 10p / mol.

[0028]

[0029]

[0030] Gel electrophoresis analysis was performed on the PCR amplification products, and the results were as follows: figure 2 shown. The results showed that the specificity of the amplified product was ...

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Abstract

The invention provides primers for detecting MPL gene mutation, and the primers include a forward primer and a backward primer for MPL gene exon 10. The invention belongs to the technical field of biological detection, and the primers provided by the invention can specifically detect MPL gene exon 10 mutation, and the specificity of the primers is good, and the accuracy is high, and the efficiency of the detection is increased.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a primer and a method for detecting MPL gene mutation. Background technique [0002] Myeloproliferative neoplasms (MPNs) are caused by hyperproliferation of multiple cell lineages resulting from alterations in growth-activating genes of hematopoietic precursor cells, including polycythemia vera (PV), essential thrombocythemia (ET), and primary Myelofibrosis (PMF). If left untreated, myeloproliferative neoplasms may eventually develop into acute myeloid leukemia. MPL gene encodes human thrombopoietin receptor (TPOR), which promotes cell growth, proliferation and differentiation. Human thrombopoietin receptor participates in the regulation of megakaryocyte proliferation and platelet production, and plays an important role in the maintenance of hematopoietic stem cells. Somatic mutations in the transmembrane region of the MPL gene were first discovered by Pikman et a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6883C12Q2535/101C12Q2565/125
Inventor 燕启江赵薇薇于世辉梁耀铭胡昌明
Owner GUANGZHOU KINGMED DIAGNOSTICS CENT
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