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Kit and method for determining mutation sites of genes of acetaldehyde dehydrogenase 2 and methylene tetrahydrofolic acid reductase by virtue of single tube at the same time

A technology of methylenetetrahydrofolate and acetaldehyde dehydrogenase, which is applied in the field of molecular biology testing and can solve the problems of increasing the cost and price of pyrosequencing testing

Inactive Publication Date: 2014-06-04
ZHONGSHAN HOSPITAL XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the amplification reaction of each site in the previous pyrosequencing needs to be carried out separately, it means that there are several mutation sites in the item to be detected, and a corresponding number of amplification reactions are required
This virtually greatly increases the cost and price of pyrosequencing as a clinical test, and passively rejects a large number of patients who need this test.

Method used

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  • Kit and method for determining mutation sites of genes of acetaldehyde dehydrogenase 2 and methylene tetrahydrofolic acid reductase by virtue of single tube at the same time
  • Kit and method for determining mutation sites of genes of acetaldehyde dehydrogenase 2 and methylene tetrahydrofolic acid reductase by virtue of single tube at the same time
  • Kit and method for determining mutation sites of genes of acetaldehyde dehydrogenase 2 and methylene tetrahydrofolic acid reductase by virtue of single tube at the same time

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The key primer components and sequences of the kit are as follows:

[0068] PCR primer 1 (Seq NO1):

[0069] ALDH2Glu504Lys (rs671) upstream primer 5'-CAGTCACCCTTTGGTGGCTACA-3';

[0070] PCR primer 2 (Seq NO2):

[0071] ALDH2Glu504Lys (rs671) downstream primer 5'-CCAGCAGGTCCCACACTCAC-3';

[0072] PCR primer 3 (Seq NO3):

[0073] MTHFR C677T (rs1801133) upstream primer 5'-GGCTGACCTGAAGCACTTGAA-3';

[0074] PCR primer 4 (Seq NO4):

[0075] MTHFR C677T (rs1801133) downstream primer 5'-CAAGTGATGCCCATGTCGGT-3'.

[0076] Sequencing Primer 1 (Seq NO5):

[0077] ALDH2Glu504Lys (rs671) sequencing primer 5'-CACACTCACAGTTTTCACTT-3';

[0078] Sequencing Primer 2 (Seq NO6):

[0079] MTHFR C677T (rs1801133) sequencing primer 5'-TGCGTGATGATGAAAT-3'.

[0080] Take 2 mL of peripheral venous blood of a healthy person A who wants to know about his ability to metabolize ethanol and folic acid, and apply the kit and method of the present invention to carry out acetaldehyde dehydrog...

Embodiment 2

[0200] Pregnant women B and C want to know their own ability to metabolize folic acid to evaluate the impact on fetal development, and pregnant woman C also wants to know the situation of their own ethanol metabolism ability, and the test kit and method of the present invention are used for detection and evaluation. 1 is exactly the same. The MTHFR (rs1801133) test results of pregnant woman B were all the same Figure 7, is G / G genotype; the MTHFR (rs1801133) test results of pregnant woman C are all as Figure 8 , is A / A genotype, the ALDH2 (rs671) test result of pregnant woman C is as follows Figure 5 It is G / G genotype; therefore, pregnant woman B has normal folic acid metabolism capacity, and a normal amount of folic acid supplementation is enough. Pregnant woman B has normal ethanol metabolism capacity, and the risk of diseases related to abnormal ALDH2 genotype is low, but her folic acid metabolism capacity is poor. Need to add more folic acid to prevent adverse effect...

Embodiment 3

[0202] Patient D with a history of myocardial infarction wants to know whether he is suitable to choose nitroglycerin as a quick-acting vasodilator emergency drug, and uses the kit and method of the present invention for detection and evaluation. The detection process is exactly the same as that of Example 1. Results The ALDH2 (rs671) test results of patient D were as follows: Figure 5 It is G / G genotype, the patient has normal metabolism ability to nitroglycerin, and nitroglycerin should be actively and effectively used for vasodilator first aid in this patient.

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Abstract

The invention provides a kit and method for determining mutation sites of genes of acetaldehyde dehydrogenase 2 and methylene tetrahydrofolic acid reductase by virtue of a single tube at the same time and belongs to the field of molecular biology test. The kit comprises a whole blood genome DNA (deoxyribonucleic acid) extraction reagent, a multiple PCR (polymerase chain reaction) amplification primer, a multiple PCR amplification reaction reagent, a single-strained DNA separation and purification reagent, a pyrosequencing primer, a pyrosequencing reagent and a kit body. The method comprises the following steps: extracting human whole blood genome DNA, carrying out multiple PCR amplification reaction, separating and purifying a single-strained DNA sample, pyrosequencing and analyzing results. The kit can be used by a person subjected to physical examination and hope to know individual ethyl alcohol and folic acid metabolic capability, can be used for predicting individual nitroglycerin metabolic capability and curative effect and guiding a patient suffering from myocardial infarction to reasonably select and prepare vasodilatation first-aid medicaments and can also be used for predicting the individual folic acid metabolic capability and guiding pregnant and lying-in women to supplement the right dosage of folic acid supplements.

Description

technical field [0001] The invention belongs to the field of molecular biology testing, in particular to a single-tube kit and method for combined determination of acetaldehyde dehydrogenase 2 (ALDH2) gene and methylenetetrahydrofolate reductase (MTHFR) gene mutation sites. The technology can be applied to clinical detection, epidemiological screening and the study of population genomic differences. In particular, it can be applied to clinical physical examination and pharmacogenomics detection. Background technique [0002] With the popularization of high-throughput sequencing and the completion of the Human Genome Project, individualized diagnosis and treatment technology is gradually moving towards daily clinical diagnosis and treatment, and is gradually affecting the way of thinking of clinical diagnosis and treatment and personal health management. Single nucleotide polymorphism (SNP) analysis is an important part of individualized diagnosis and treatment. Currently, i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/6869C12Q2600/106C12Q2600/156
Inventor 叶辉铭苏晓崧张忠英
Owner ZHONGSHAN HOSPITAL XIAMEN UNIV
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