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Fecal DNA quantitative methylation specific PCR detection kit and applications thereof

A kit and methylation technology, applied in the field of epimutation analysis and consequence assessment, can solve problems such as low transcriptional activity, abnormal expression of related proteins, and related gene silencing

Inactive Publication Date: 2015-06-10
NANJING HOSPITAL OF T C M
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Problems solved by technology

[0003] It is known that the epimutation of tumor suppressor genes is an early event in the occurrence of cancer. Abnormal methylation of the DNA promoter region can lead to silencing or low transcriptional activity of related genes, resulting in abnormal expression of related proteins and triggering cancer

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  • Fecal DNA quantitative methylation specific PCR detection kit and applications thereof
  • Fecal DNA quantitative methylation specific PCR detection kit and applications thereof
  • Fecal DNA quantitative methylation specific PCR detection kit and applications thereof

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Embodiment 1

[0039] Example 1: Analysis of the methylation status of CpG islands in the promoter regions of SPG20, SNCA and FBN1 genes in Chinese colorectal cancer patients and normal controls: 11 and 19 Chinese colorectal cancer patients and normal controls respectively collected feces, separated and extracted the shedding in the feces Intestinal mucosa, intestinal mucosal DNA was extracted. According to CHEMICON CpGenome TM DNA modification kit instruction manual operation steps for DNA bisulfite modification. The modified DNA was subjected to quantitative methylation-specific PCR (qMSP), and the primers and probes are shown in Table 1. The reaction system is 10μl: TaKaRa Premix Ex Taq TM Polymerase 5 μl, upstream primer (0.9 μM) 0.9 μl, downstream primer (0.9 μM) 0.9 μl, TaqMan probe (3 μM) 0.4 μl, bisulfite-modified DNA (200 ng / μl) 2.6 μl, ROX Ref 0.2 μl. PCR reaction conditions: pre-denaturation at 95°C for 30 sec; denaturation at 95°C for 10 sec, refolding at around 60°C for 30 se...

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Abstract

The invention relates to a fecal DNA quantitative methylation specific PCR detection kit and applications thereof. The kit comprises (1) three pairs of amplification primers for the quantitative methylation of colon tumor incidence related CpG-islands in a cancer suppressor gene promoter region and three probes; and (2) a PCR reagent for amplification. A PCR amplification product obtained through the kit can analyze the methylation status of gene CpG-islands through a (quantitative methylation specific PCR, qMSP) PCR method. The kit has an efficient CpG-island methylation detection effect. The kit is applicable to the epigenetic mutation detection of colon tumor incidence related cancer suppressor genes in faeces.

Description

technical field [0001] The invention relates to a kit for detecting the epimutation of a tumor suppressor gene related to the onset of colon tumors in feces, in particular to the analysis of the epimutation and the evaluation of its consequences in Chinese colon cancer patients. Background technique [0002] Colorectal cancer is the second and third most common malignant tumor in my country's large and medium-sized cities, and the effect of early detection and early treatment is ideal. Therefore, it is particularly important to find a non-invasive early detection method with high sensitivity and specificity. [0003] It is known that the epimutation of tumor suppressor genes is an early event in the occurrence of cancer. Abnormal methylation of the DNA promoter region can lead to silencing or low transcriptional activity of related genes, resulting in abnormal expression of related proteins and triggering cancer. The study found that the methylation levels of CpG island reg...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/154
Inventor 金黑鹰范怡梅杨青蓝邓超
Owner NANJING HOSPITAL OF T C M
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