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Primers for quantitatively detecting methylation level of rat H19 gene imprinting control region by sodium bisulfite sequencing process

A sodium bisulfite, genetic imprinting technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of inability to develop molecular genetics laboratories and expensive instruments

Inactive Publication Date: 2016-04-20
ZHEJIANG ACAD OF MEDICAL SCI
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Problems solved by technology

However, the equipment is expensive and cannot be carried out in ordinary molecular genetics laboratories

Method used

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  • Primers for quantitatively detecting methylation level of rat H19 gene imprinting control region by sodium bisulfite sequencing process
  • Primers for quantitatively detecting methylation level of rat H19 gene imprinting control region by sodium bisulfite sequencing process
  • Primers for quantitatively detecting methylation level of rat H19 gene imprinting control region by sodium bisulfite sequencing process

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Embodiment Construction

[0019] The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.

[0020] 1. Analysis of imprinted control region and primer design

[0021] p,p'-DDE exposed rat sperm count and motility significantly decreased, imprinted gene H19 (the gene GenBankAccession is NC_005100.3) expression was significantly up-regulated, in order to test the accuracy of the screening results, the present invention controls the gene imprinted region Perform CpG island analysis such as figure 1 shown. Using bisulfite-converted genomic DNA as a template, sodium bisulfite PCR primers were designed using methprimer software and synthesized by Invitrogen. Primer sequences are shown in Table 1.

[0022] Table 1

[0023]

[0024] 2. Rat sperm cell genomic DNA extraction

[0025] Collect sperm cells from 6 rats (3 rats are the p,p'-DDE exposure group and 3 rats are the control group), and use a genomic DNA extraction kit to...

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Abstract

The invention discloses primers for quantitatively detecting methylation level of a rat H19 gene imprinting control region by a sodium bisulfite sequencing process. The primers comprise PCR (polymerase chain reaction) primers and sequencing primers, wherein the sequences of the forward primer of the PCR primers and the forward primer of the sequencing primers are respectively 5'-AGTGTGTAGGGATTAAGGGGAAATT-3'; and the sequences of the reverse primer of the PCR primers and the reverse primer of the sequencing primers are respectively 5'-TCAATCTCAATTACAATCTATTTCAACAAA-3'. The primers are used for screening the H19 gene imprinting control region in a p,p'-DDE exposed rat; the PCR primers and sequencing primers are designed according to the imprinting control region; and sodium bisulfite sequencing can be performed to carry out accurate quantitative detection on the methylation level of the imprinting control region.

Description

technical field [0001] The invention relates to the field of gene methylation detection, in particular to a primer for quantitatively detecting the methylation level of the imprinted gene H19 imprinted control region by a sodium bisulfite sequencing method. Background technique [0002] Genomic imprinting is a phenomenon of differential expression of parental alleles that does not follow Mendelian inheritance rules. The asymmetric expression of alleles is controlled by modifying the imprinting control region near the promoter. This modification is often DNA methylation modification also includes histone acetylation, methylation and other modifications. In the study of gene imprinting and male reproduction, it was found that imprinted genes were abnormally methylated, resulting in altered expression. Therefore, gene imprinting is closely related to male reproduction, and the study of imprinted gene methylation can not only provide an important basis for revealing the mechani...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/158
Inventor 宋杨吴南翔高明王禹范宏亮谭玉凤姚春冀刘克澄
Owner ZHEJIANG ACAD OF MEDICAL SCI
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