Primer, probe, kit and method for detection of human ALDH2 gene polymorphism

A gene polymorphism and kit technology, applied in the field of genetic engineering, can solve the problems of inability to accurately locate gene changes, expensive chips, inability to accurately detect gene polymorphisms in samples, etc., and achieve intuitive and convenient result interpretation and fluorescence analysis. Accurate analysis and the effect of reducing background signal strength

Inactive Publication Date: 2017-07-14
SHANGHAI TISSUEBANK MEDICAL LAB CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the sequencing method is the "gold standard" for gene polymorphism detection, it requires a long detection time, complicated operation, and low sensitivity; the PCR-gene chip method has high operational requirements, and hybridization and washing must be protected from light operation, and the chip is expensive; the PCR-melting curve method is non-specific and cannot precisely locate the position of the gene change, and the difference in Tm caused by a single base difference is not very obvious, which usually results in inconspicuous peaks on the melting curve Therefore, there are certain difficulties in the clini...

Method used

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  • Primer, probe, kit and method for detection of human ALDH2 gene polymorphism
  • Primer, probe, kit and method for detection of human ALDH2 gene polymorphism
  • Primer, probe, kit and method for detection of human ALDH2 gene polymorphism

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1: Using cell line DNA samples containing polymorphic sites of ALDH2 genes to verify the specificity of the detection system of the present invention

[0041] In this example, the feasibility and specificity of the detection method of the present invention are verified by determining the ALDH2-GG type H2228 cell line, the ALDH2-AA type Kasumi cell line, and the ALDH2-GA type Kasumi and H2228 heterozygous cell line through sequencing.

[0042] The specific detection method is as follows:

[0043] Using the PCR reaction system and reaction conditions described in this manual, using Kasumi DNA, H2228 DNA, and the mixed DNA of Kasumi and H2228 as templates, each DNA sample was spotted once for detection.

[0044] The genotyping results of the test are shown in the attached instructions figure 1 shown. Among them, NTC is the negative control, the H2228 DNA sample well is ALDH2-GG type, the Kasumi DNA sample well is ALDH2-AA type, and the mixed DNA sample well of Kas...

Embodiment 2

[0046] Example 2: Using human genomic DNA samples to detect ALDH2 gene polymorphisms to verify the repeatability of the detection system of the present invention

[0047] In this example, human genomic DNA samples were used as templates for fluorescent PCR genotyping detection, and the detection results were directly compared with the "gold standard" sequencing results to verify the repeatability and accuracy of the detection system of the present invention.

[0048] The specific detection method is as follows:

[0049] Using the PCR reaction system and reaction conditions described in this manual, the human genome DNA sample is used as a template for fluorescent PCR genotyping detection.

[0050] The genotyping results of 78 representative human genomic DNA samples are shown in the attached instructions Figure 2A As shown, where NTC is the negative control, except for the positive control, 47 samples are ALDH2-GG homozygous, 28 samples are ALDH2-GA heterozygous, and 3 sampl...

Embodiment 3

[0052] Example 3: Using Human Genomic DNA Samples to Detect ALDH2 Gene Polymorphisms to Verify the Sensitivity of the Detection System of the Present Invention

[0053] In this embodiment, the sensitivity of the detection system of the present invention is also verified by using the human genome DNA sample as a template.

[0054] The specific detection method is as follows:

[0055] Using the PCR reaction system and reaction conditions described in this manual, select 7 cases of human genomic DNA samples as templates for fluorescent PCR genotyping detection, and set the detection limit groups of each DNA template as follows: 0.05ng; 0.1ng; 0.5ng ;1ng;10ng;50ng;100ng. The results show that clear genotyping results can still be obtained under the condition that the amount of DNA template is only 1ng. The representative genotyping results are shown in the attached instructions. image 3 As shown, NTC is the negative control. In addition to the positive control, 5 samples are AL...

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Abstract

The invention belongs to the technical field of gene engineering and discloses a primer and a probe combination for detection of human ALDH2 gene polymorphism, a kit comprising the primer and the probe combination, and a fluorescent PCR method for performing the detection of human ALDH2 gene polymorphism with the primer and the probe combination. The primer, the probe and the kit are based on TaqMan fluorescent PCR technology, and are simple, quick and high-sensitive. In addition, through reasonable combination of primer and probe, interaction between primer and primer, primer and probe and probe and probe can be avoided effectively, thus reducing detection error. The primer, the probe and the kit, when being used for the detection of human ALDH2 gene polymorphism, have high sensitivity and strong specificity, are easy, quick and safe to operate, have simple and direct results, and can be applied directly with a blood sample or a dried blood spot specimen on filter paper as a template.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to primers, probes, kits and methods for detecting human ALDH2 gene polymorphisms. Background technique [0002] Nitroglycerin is an emergency drug for the prevention and treatment of coronary heart disease and angina. The enzyme promotes calcium ions to enter the sarcoplasmic reticulum and the extracellular space, thereby dilating vascular smooth muscle and relieving the symptoms of cardiac ischemia. The polymorphism of ALDH2*2 (Glu504Lys, rs671) leads to the substitution of glutamic acid at position 504 of the encoded protein by lysine, and the ALDH2 enzyme activity of individuals carrying the mutant allele ALDH2*2 decreases, and the enzyme activity of heterozygotes is only wild type In 10% of individuals, the mutation is homozygous for loss of enzyme activity. Therefore, patients with ALDH2*2 type cannot use nitroglycerin to resist myocardial ischemia. The carrier ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/106C12Q2600/156C12Q2561/101C12Q2563/107
Inventor 郑仲征安琳芮立尤开
Owner SHANGHAI TISSUEBANK MEDICAL LAB CO LTD
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