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Detection method and detection kit of gene locus types of DNA (Deoxyribose Nucleic Acid) methyltransferase 3 alpha

A methyltransferase and detection kit technology, which is applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of not developing multi-gene locus detection technology and method, and achieve great clinical application value. , Simple operation, the effect of reducing the detection time

Inactive Publication Date: 2014-08-20
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection technology and method for multi-gene loci have not been developed yet

Method used

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  • Detection method and detection kit of gene locus types of DNA (Deoxyribose Nucleic Acid) methyltransferase 3 alpha
  • Detection method and detection kit of gene locus types of DNA (Deoxyribose Nucleic Acid) methyltransferase 3 alpha
  • Detection method and detection kit of gene locus types of DNA (Deoxyribose Nucleic Acid) methyltransferase 3 alpha

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The steps of the detection method for DNMT3α gene locus typing are as follows:

[0036] 1 Genomic DNA extraction

[0037] The peripheral blood of acute leukemia without DNMT3α gene mutation was extracted, and extracted according to the operating instructions of Promaga Genomic DNA Extraction Kit.

[0038] 2 PCR amplification

[0039] The extracted DNA was amplified by PCR: the PCR primers used were two pairs of forward primers and reverse primers,

[0040] Forward primer 5'-TCCTGCTGTGTGGTTAGACG-3'

[0041] Reverse primer 5'-CCATGTCCCTTACACACACG-3'

[0042] The components of the PCR system are shown in Table 1:

[0043] Table 1

[0044] Reagent concentration system wxya 2 o 9.1 polymerase buffer 10× 2 Mgcl 2 50mM 2.4 dNTP 10mM 2 polymerase 2U / μL 0.5 sample 20-50ng / μL 2 Primer 2mM 2

[0045] PCR reaction program: denaturation and enzyme activation at 95°C for 15 minutes, denaturation at 94°C...

Embodiment 2

[0058] In Example 1, the peripheral blood genomic DNA of the acute leukemia DNMT3α gene mutation case was replaced with the peripheral blood genomic DNA of the acute leukemia DNMT3α gene mutation case, and the others were the same. See the test results figure 2 .

Embodiment 3

[0060] In Example 1, the peripheral blood genomic DNA of the acute leukemia DNMT3α gene non-mutated case was replaced with the peripheral blood genomic DNA of the acute leukemia DNMT3α gene heterozygous case, and the others were the same. See the test results image 3 .

[0061] From Figure 1-3 It can be analyzed that:

[0062] sample DNMT3α sample 1 G G sample 2 A A sample 3 G A

[0063] It can be seen from the figure that sample 1 has a single peak at 75bp, and the DNMT3α genotype is GG; sample 2 has a single peak at 77bp, and the DNMT3α genotype is AA; sample 3 has double peaks at 75bp and 77bp, and the DNMT3α genotype for GA.

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Abstract

The invention discloses a detection method and a detection kit of gene locus types of DNA (Deoxyribose Nucleic Acid) methyltransferase 3 alpha. The method comprises the following steps of performing PCR (Polymerase Chain Reaction) amplification on genome DNA as a template to obtain a PCR amplification product; performing LDR (Ligase Detection Reaction) amplification on the PCR amplification product to obtain an LDR amplification product; analyzing the LDR amplification product by using the Genescan function of a sequenator, judging gene types of drug-resistant loci, and providing information of specific drug-related loci. According to the detection method and the detection kit, various gene types of DNMT3 alpha can be conveniently detected by adopting the PCR-LDR method; the detection method has the characteristics of being reliable and stable in result, simple to operate and quick; complicated steps of purifying the product, depositing and the like in a conventional PCR sequencing detection method are removed, the detection time is shortened, and a relatively high clinical application value is achieved.

Description

technical field [0001] The invention relates to a detection method of acute myeloid leukemia mutation gene, in particular to a DNA methyltransferase 3α (DNMT3α) gene site typing detection method and a detection kit. Background technique [0002] Acute myeloid leukemia is one of the most serious diseases that harm young people. DNMT3α mutation provides a new molecular biological marker for the prognosis of acute myeloid leukemia, especially the R882 site is a mutation hotspot. [0003] Ligase detection reaction (LDR, ligase detection reaction) is a nucleic acid detection technology developed in recent years. Only a pair of probes are added to the reaction, and the template is linearly amplified. It is mainly used in the field of single nucleotide detection, such as single nucleotide polymorphism detection (SNP) (Detection of HLA Polymorphisms by Ligase Detection Reaction and a Universal Array Format: A Pilot Study for Low Resolution Genotyping. Clarissa Consolandi, Elena Bust...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2561/125C12Q2531/113C12Q2545/101
Inventor 祁小飞
Owner SUZHOU UNIV
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