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Serine hydroxymethyltransferase gene promoter region methylation degree detection kit and method

A technology of serine hydroxymethyl group and gene promoter region is applied in the field of detection kits for the methylation degree of serine hydroxymethyltransferase gene promoter region, which can solve the problems of less research on the regulation mechanism of homocysteine ​​and the like, Achieve the effect of low detection cost, strong specificity, and ensure accuracy

Inactive Publication Date: 2018-03-27
深圳市南山区慢性病防治院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Now domestic and foreign studies have confirmed that the level of homocysteine ​​is closely related to the occurrence of stroke, but there are few studies on the factors affecting the level of homocysteine ​​and its regulation mechanism

Method used

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  • Serine hydroxymethyltransferase gene promoter region methylation degree detection kit and method
  • Serine hydroxymethyltransferase gene promoter region methylation degree detection kit and method
  • Serine hydroxymethyltransferase gene promoter region methylation degree detection kit and method

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Effect test

Embodiment 1

[0054] A detection kit for the degree of methylation in the promoter region of the serine hydroxymethyltransferase gene, the detection kit includes a DNA extract, a fluorescent quantitative PCR reaction solution, a positive control, a negative control, and a plurality of sealed tubes with caps. Fluorescent quantitative PCR reaction solution includes the PCR reaction solution as 10×PCR buffer 2μL, 25mmol / L MgCl 2 1.5μL, 2.5mmol / L d NTPs 1.5μL, 10μM Primer F 0.25μL, 10μM Primer R 0.25μL, 5U / μL hot-start Taq DNase 0.2μL, DNA template 1μL, 2×SYBR Green I 5μL, sterile double-distilled Make up to 20 μL with water; the nucleotide sequences of Primer F and Primer R are as follows:

[0055] Primer F: 5'-CGAGTTTAGGAAGGTGTATT-3' (SEQ ID NO.1) and

[0056] Primer R: 5'-CCATACTTAACTACGCTCTC-3' (SEQ ID NO.2);

[0057] The positive control is a complete DNA methylation sample obtained after the genome of a normal human sample is completely methylated; the negative control is sterile double...

Embodiment 2

[0059] A method for determining the degree of methylation in the promoter region of a serine hydroxymethyltransferase gene, comprising:

[0060] DNA extraction steps: use Lab-Aid 820 automatic nucleic acid extractor to extract whole blood genomic DNA and use NanoDrop2000 ultra-micro spectrophotometer to detect the concentration of DNA;

[0061] Using a methylation kit to perform bisulfite conversion on whole blood genomic DNA Step: Using the EZ DNAMethylation-Gold kit to react the above-mentioned extracted whole blood genomic DNA with bisulfite will make the unmethylated DNA in the single strand Cytosine is converted to thymine, and then converted DNA is obtained through steps such as loading, washing, and elution;

[0062] DNA methylation level determination steps: the above-mentioned converted DNA is used as sample DNA and added to the reaction solution containing fluorescent quantitative PCR for PCR amplification reaction, so that the gene sequence is multiplied. Each sampl...

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Abstract

The invention discloses a serine hydroxymethyltransferase gene promoter region methylation degree detection kit. The detection kit comprises DNA extract, a fluorescence quantitative PCR reaction solution, a positive control, a negative control and a plurality of seal pipes with covers. The fluorescence quantitative PCR reaction solution comprises a pair of serine hydroxymethyltransferase gene promoter region methylation specific amplification primers shown in the formulas of SEQ ID NO. 1 and SEQ ID NO. 2. The invention also discloses a method for detecting a serine hydroxymethyltransferase gene promoter region methylation degree. The kit utilizes a fluorescence quantitative PCR technology, has the advantages of real-time monitoring, strong specificity and accurate quantification and ensures the accuracy of the diagnosis result.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection kit and a determination method for the degree of methylation in the promoter region of a serine hydroxymethyltransferase gene. Background technique [0002] Cerebral stroke is an acute cerebrovascular disease, which is a group of diseases that cause brain tissue damage due to the sudden rupture of blood vessels in the brain or the inability of blood to flow into the brain due to vascular obstruction, including ischemic and hemorrhagic strokes. Stroke has the characteristics of high morbidity, high mortality and high disability rate. The clinical manifestations are severe headache, fainting, loss of consciousness, etc. It has also become the leading cause of disability in Chinese adults. Stroke patients need to take antihypertensive drugs for a long time, conduct regular routine examinations, and control intracranial pressure. It can be seen that stroke patient...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6851
CPCC12Q1/6851C12Q1/6883C12Q2600/154C12Q2531/113C12Q2563/107C12Q2545/101
Inventor 王长义彭晓琳张敏马剑平温琪张涛
Owner 深圳市南山区慢性病防治院
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