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Methylated amplification method for eliminating PCR product pollution and application of methylated amplification method

A methylation and product technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems that UDG enzymes cannot be used, polluted, and it is difficult to solve methylation amplification PCR products, etc.

Active Publication Date: 2017-12-19
GENE TECH SHANGHAI COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Therefore, the uracil contained in the bisulfite conversion product makes the UDG enzyme unable to be used in the methylation amplification system. At present, it is difficult to solve the pollution problem of the methylation amplification PCR product in this field.

Method used

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  • Methylated amplification method for eliminating PCR product pollution and application of methylated amplification method
  • Methylated amplification method for eliminating PCR product pollution and application of methylated amplification method
  • Methylated amplification method for eliminating PCR product pollution and application of methylated amplification method

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Embodiment 1

[0067] Embodiment 1, the methylation amplification example of MGMT gene

[0068] In the present embodiment, detect O with the method of the present invention 6 - Methylation of the methylguanine-DNA-methyltransferase (MGMT) gene. The MGMT gene is located at 10q26 on chromosome 10, and its function is to convert O 6 - Guanine adducts are removed from DNA and damaged DNA is repaired. When the CpG site in the promoter region of the MGMT gene is methylated, the transcription of MGMT stops and Guanine-O 6 The failure of the transfer of methyl groups on DNA leads to the inability to effectively repair DNA alkylation damage, which is closely related to tumorigenesis. The results of the study showed that: MGMT promoter methylation is closely related to the clinical effect of the first-line chemotherapy drug Temozolomide. It is generally believed that tumor cells with MGMT methylation are effective against alkylating agents, while non-methylation of MGMT gene means resistance to al...

Embodiment 2

[0098] Example 2, the application example of eliminating PCR product contamination in the detection of MGMT gene methylation

[0099] The PCR amplification method of the present invention eliminates the PCR product contamination problem in the MGMT gene methylation amplification system.

[0100] 1. Test sample

[0101] The test samples were MGMT methylation-negative samples (HT29 cell line DNA) and MGMT methylation-positive samples (SW480 cell line DNA). The concentration is 10ng / μL, which is used as DNA template for PCR amplification.

[0102] PCR amplification contaminants are amplification products of methylation-positive cell line (SW480), which are purified and diluted to a final concentration of 10,000 copies / microliter.

[0103] Four kinds of samples containing or not containing PCR amplification pollutants were prepared, see Table 1 for details.

[0104] Table 1

[0105]

[0106] 2. PCR amplification system

[0107] Two fluorescent PCR systems were prepared, th...

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Abstract

The invention relates to a methylated amplification method for eliminating PCR product pollution and application of the methylated amplification method. Sub-system and sub-step heat treatment is carried out before a PCR reaction, so that uracil-DNA-glycosylase is effectively applied, and the pollution problem of methylated amplification PCR products is effectively solved.

Description

technical field [0001] The invention belongs to the field of gene detection. More specifically, the invention relates to a methylation amplification method capable of effectively eliminating PCR product pollution and its application. Background technique [0002] DNA methylation is one of the ways of gene epigenetic modification, which may exist in all higher organisms. DNA methylation shuts down the activity of certain genes, while demethylation induces reactivation and expression of genes. In mammals, DNA methylation only occurs on CpG cytosine, and under the action of DNA methyltransferases (DNMTs), the cytosine in CpG dinucleotides is converted into 5-methylcytosine ( m C). This DNA modification does not change the gene sequence, but it can regulate gene expression. DNA methylation is the most well-studied epigenetic mechanism, which plays an important role in the maintenance of normal cell function, modification of chromatin structure, X chromosome inactivation, geno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6848C12Q1/6858C12Q2531/113C12Q2521/531C12Q2527/125
Inventor 王东李宾赵静钮琪
Owner GENE TECH SHANGHAI COMPANY
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