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Kit for detecting thiopurine methyltransferase genotype, method and application thereof

A mercaptopurine methyl and kit technology, applied in the biological field, can solve the problems of treatment failure, treatment interruption, and high disease recurrence rate, and achieve the effects of improving amplification efficiency and saving time.

Inactive Publication Date: 2009-12-02
BEIJING HUAANFO BIOMEDICAL RES CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Individuals with high TPMT activity have a low concentration of active metabolites-mercaptoguanine phosphates (TGNs) after medication, so the recurrence rate of the disease is high; and for individuals with TPMT deficiency, medical conventional doses of purine drugs can also cause in vivo The accumulation of mercaptoguanine phosphate produces serious side effects such as severe bone marrow suppression and liver damage, which eventually leads to the forced interruption of treatment, resulting in treatment failure (Coulthard SA, Howell C, Robson J, et al.Blood, 1998 , 92: 2856-2862; Relling M V, Hancock ML, Boyett JM, et al. Blood, 1999, 93: 2817-2823)

Method used

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  • Kit for detecting thiopurine methyltransferase genotype, method and application thereof
  • Kit for detecting thiopurine methyltransferase genotype, method and application thereof
  • Kit for detecting thiopurine methyltransferase genotype, method and application thereof

Examples

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Embodiment 1

[0076] The present invention will be further described in detail below by taking the TPMT G460A wild-type positive control plasmid as an example.

[0077] 1. Primers and templates

[0078] Gene fragment amplification uses the following primers:

[0079] TPMT 460 For: 5'-CAGCGTAGTTGGTGTGGAAA-3';

[0080] TPMT 460 Back: 5'-GCGATCACCTGGATTAATGG-3'.

[0081] The template uses human genomic DNA (No. AI011611, concentration 15ng / μl)

[0082] 2. Gene fragment amplification

[0083] To configure the PCR amplification mixture:

[0084] Template (1515ng / μl): 3μl;

[0085] 10×PCR buffer (-MgCl 2 ): 1 μl;

[0086] 50mM MgCl 2 : 0.3 μl;

[0087] 10mM dNTPs: 0.2μl;

[0088] 20 μM primer mix: 0.2 μl;

[0089] 5U / μl Taq enzyme: 0.05μl;

[0090] Sterile double distilled water: 5.25 μl;

[0091] Total: 10 μl.

[0092] _PCR amplification conditions are:

[0093] 94°C for 3 minutes; 94°C for 30 seconds, 63°C for 45 seconds, 72°C for 30 seconds, 35 cycles; 72°C for 7 minutes.

[00...

Embodiment 2

[0125] The present invention will be further described in detail below by taking the TPMT genotype detection process of leukemia patient genes as an example.

[0126] We used this kit to detect the mercaptopurine methyltransferase genotypes of 20 patients, the process is as follows:

[0127] 1. Sample processing:

[0128] Since the sample is genomic DNA, use a UV spectrophotometer to detect the DNA concentration of the sample, and dilute the DNA concentration to 30-100ng / ml

[0129] 2. Configuration of G460A-specific PCR amplification mixture

[0130] Configure G460A amplification mixture: PCR reaction mixture: 2.5ul;

[0131] G460A primer + probe: 1ul;

[0132] H2O: 6.5ul;

[0133] Total: 10ul.

[0134] Configure A719G amplification mixture: PCR reaction mixture: 2.5ul;

[0135] A719G primer + probe: 1ul;

[0136] H2O: 6.5ul;

[0137] Total: 10...

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Abstract

The invention belongs to the gene technical field and relates to a kit for detecting thiopurine methyltransferase genotype, a method and an application thereof. In the invention, the single nucleotide polymorphism Genotyping method of fluorescent quantitative PCR is adopted; in the method, auele specific primers of thiopurine methyltransferase gene polymorphism locus and fluorescently-labeled probes are adopted to carry out polymerase chain reaction on sample DNA, carry out fluorescence scanning and classify the genotype according to fluorescence types; wherein the primers comprise the auele specific primers for detecting thiopurine methyltransferase G460A polymorphism locus and the auele specific primers for detecting A719G polymorphism locus. The probes comprise a thiopurine methyltransferase G460A polymorphism locus wild type specific probe and a mutant type specific probe and a wild type specific probe and a mutant type specific probe for detecting A719G polymorphism locus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a mercaptopurine methyltransferase genotype detection kit, method and application. More specifically, the present invention relates to a kit, method and application for analyzing the polymorphism of a gene locus encoding mercaptopurine methyltransferase by polymerase chain reaction method. Background technique [0002] Purine drugs, including 6-mercaptopurine (6-MP), 6-mercaptopurine (6-TG) and mercaptopurine (AZA) are commonly used clinical tumor chemotherapy drugs. They interfere with the metabolism of nucleic acids in the body, including Both de novo and salvage synthesis pathways of nucleic acids come into play. Purine drugs are drugs without intrinsic biological activity, and must undergo a series of metabolism in the body before they can exert their anti-leukemic effect. [0003] Studies have shown that the efficacy of purine drugs is closely related to the activity of thiopuri...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 张彦明于多王燕徐希平
Owner BEIJING HUAANFO BIOMEDICAL RES CENT
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