Method for screening heterozygote during allele specific methylation
An allele-specific and methylation technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem of hindering the combination of transcription factors and promoters, uncontrolled synthesis of mature functional proteins, cell proliferation, and gene transcription disorders. Live and other issues
Inactive Publication Date: 2014-02-19
王景文
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Problems solved by technology
Methylation of the promoter region of tumor suppressor genes can hinder the binding of transcription factors to the promoter, resulting in inactivation of gene transcription, blocking the synthesis of mature functional proteins, and leading to uncontrolled cell proliferation
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[0009] A method for the detection of heterozygotes in allele-specific methylation. Specifically, after the DNA is cut with a methylation-sensitive restriction endonuclease, PCR amplification is performed with specific primers containing the sequence of the restriction endonuclease. By detecting the PCR amplification products of methylation-sensitive enzymes, the methylation status of the two alleles can be detected; MSRE-PCR detection is performed to identify whether there is a difference in the methylation status of specific sites of biallelic genes; according to Methylation-sensitive restriction endonucleases can only recognize and cut off unmethylated cleavage sites, but cannot recognize and cut off methylated DNA sequences.
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The invention discloses a method for screening a heterozygote during allele specific methylation. Specifically, the method comprises: performing methylation sensitivity restriction enzyme digestion on DNA, performing PCR amplification by employing a specific primer containing an enzyme digestion site sequence; detecting PCR amplification product of methylation sensitive enzyme to detect the methylation state of two alleles; performing MSRE-PCR detection to determine whether the methylation state of two allele specific sites possesses difference. The principle as the base is that methylation sensitive restriction enzyme only helps to identify and digest enzyme digestion sites not subjected to methylation, and cannot help to identify and digest a DNA sequence subjected to methylation. The beneficial effects comprise that: the method helps to effectively prevent a transcription factor from combining with a promoter, lead transcription inactivation, and further lead cell proliferation control loss because of synthetic obstruction of mature functional protein.
Description
technical field [0001] The invention relates to a biochemical detection method, in particular to a method for detecting heterozygotes in allele-specific methylation. Background technique [0002] DNA methylation is one of the most important epigenetic modifications and is closely related to tumorigenesis. Methylation of the promoter region of tumor suppressor genes can hinder the combination of transcription factors and promoters, resulting in inactivation of gene transcription, blocking the synthesis of mature functional proteins and resulting in uncontrolled cell proliferation. The research on methylation inactivation of tumor suppressor gene has important guiding significance in the early diagnosis, treatment and prognosis of clinical tumors, and is a current research hotspot. [0003] We previously tested the methylation of multiple sites in gastric cancer in the high-incidence area in southern Liaoning, and found that patients with gastric cancer in this area P16 , h...
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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2521/301
Inventor 王景文
Owner 王景文