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Primer for detecting methylation level of GPVI gene promoter region and detection method thereof

A gene promoter region and detection method technology, applied in the detection field of primers and its detection of the methylation level of the GPVI gene promoter region, can solve the problems of structure shrinkage, target gene expression reduction, unfavorable transcription initiation, etc.

Pending Publication Date: 2020-04-03
HENAN UNIV OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

When the CpG site is in a methylated state, it will lead to changes in the conformation of DNA in certain regions, causing its structure to shrink and the helix to deepen, thereby affecting the interaction of transcription factors or coactivators with related regulatory regions on DNA, which is not conducive to transcription Initiation of the target gene, resulting in decreased expression of the target gene, manifested as a silencing state

Method used

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  • Primer for detecting methylation level of GPVI gene promoter region and detection method thereof
  • Primer for detecting methylation level of GPVI gene promoter region and detection method thereof
  • Primer for detecting methylation level of GPVI gene promoter region and detection method thereof

Examples

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Embodiment 1

[0060] A primer for detecting the methylation level of the GPVI gene promoter region, including a pair of amplification primers and a sequencing primer GPVI-S (as shown in SEQ ID NO: 3) of the GPVI gene promoter region after bisulfite conversion;

[0061] The amplification primer pair comprises upstream primer GPVI-F (shown in SEQ ID NO:1) and downstream primer GPVI-R (shown in SEQ ID NO:2),

[0062] The sequence of the upstream primer GPVI-F is 5'-ATTAGGGAGTTTATGGGAGTACGG-3', the sequence of the downstream primer GPVI-R is: 5'-Biotin-ATTCCTCAACCCCTATCCTAAACTCTAT-3'; the sequence of the sequencing primer GPVI-S is: 5'-AATATAGATTAGGTTTTAGTAGG-3'.

Embodiment 2

[0064] A detection method for detecting the methylation level of the GPVI gene promoter region, using the amplification primer pair described in Example 1 to amplify the human blood GPVI gene promoter region after bisulfite conversion, and using the described The sequencing primer GPVI-S detects the methylation ratio of the 5 CpG sites in the detected gene region, and the expression level of the GPVI gene in blood is determined by the methylation rate obtained through the detection.

[0065] Specifically include the following steps:

[0066] (1) Use EDTA anticoagulant blood collection tubes to collect 2ml of venous blood from 31 cases of clinical samples CHD and 31 cases of healthy group;

[0067] (2) Separate the leukocytes in the blood of 31 cases of clinical samples CHD collected in step (1) and 31 cases of healthy groups by Ficoll method;

[0068] (3) Genomic DNA in white blood cells was extracted by column extraction method, and genomic DNA of white blood cells was extra...

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Abstract

The invention discloses a primer for detecting methylation level of a GPVI gene promoter region and a detection method of the primer. The primer comprises an amplification primer pair of the GPVI genepromoter region after bisulfite conversion and a sequencing primer GPVI-S, the sequence of the upstream primer GPVI-F is 5'-ATTAGGGAGTTTATGGGAGTACGG-3', and the sequence of the downstream primer GPVI-R is 5'-Biotin-ATTCCTCAACCCTATCCTAAACTCTAT-3'; the sequence of the sequencing primer GPVI-S is 5'-AATATAGATTAGGTTTTAGTAGG-3'. A pyrosequencing method is adopted for sequencing, the methylation and non-methylation proportions of a CpG island in a target region are efficiently detected, the methylation level of the GPVI promoter region is evaluated, and the methylation level can be used as an earlyindex for myocardial infarction.

Description

technical field [0001] The invention relates to the technical field of biological gene detection. Specifically, it is a primer for detecting the methylation level of the GPVI gene promoter region and a detection method thereof. Background technique [0002] Overactivation of platelets can lead to unnecessary platelet aggregation, eventually leading to thrombosis and cardiovascular disease. In recent years, many studies have shown that the occurrence and development of coronary heart disease are closely related to the disturbance of epigenetic DNA methylation patterns. Therefore, studying the mechanism of DNA methylation in the pathogenesis of coronary heart disease can provide new ideas for the diagnosis and treatment of coronary heart disease. [0003] When platelets participate in the development of coronary heart disease, the receptors on the platelet membrane play a key role. Glycoprotein VI (GPVI) is one of the many receptors on the platelet membrane, and it is also ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6869C12N15/11
CPCC12Q1/6883C12Q1/6869C12Q2600/154C12Q2523/125C12Q2531/113C12Q2565/301
Inventor 吴鸿韩勇军高水波王振涛雷震王新洲高海霞
Owner HENAN UNIV OF CHINESE MEDICINE
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