Methylation quantitative detection method of APC gene in human plasma

A technology of methylation quantification and detection method, applied in the field of biomacromolecule detection, can solve the problems of detection difficulties, early diagnosis, prognosis and recurrence monitoring of tumor patients without direct application, and achieve the effect of improving detection sensitivity

Inactive Publication Date: 2009-01-28
潘世扬
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Problems solved by technology

Due to the low DNA content in plasma, the high loss rate during non-standardized extraction, the large amount of DNA degradation during non-standardized bisulfite chemical modification, and the low sensitivity and carcinogenic pollutant ethidium bromide after common MSP Due to the electrophoresis results and other reasons, it i...

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  • Methylation quantitative detection method of APC gene in human plasma
  • Methylation quantitative detection method of APC gene in human plasma

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Embodiment Construction

[0029] 1. Plasma separation and storage

[0030] with EDTA-K 2 Extract 2ml of human peripheral venous blood from a 5ml sterile plastic anticoagulant tube with a cover, and centrifuge at 3000rpm for 10min at room temperature for 48 hours to collect plasma; centrifuge again at 12000rpm for 10min to obtain blood cell-free plasma The tubes are divided into 200 μl per tube, stored at a temperature below -20°C, and tested within one month.

[0031] Note: Glass centrifuge tubes cannot be used to collect anticoagulated venous blood.

[0032] 2. Standard Curve Plasma Sample Preparation

[0033] (1) Human fetal cord blood (CB) cell DNA extraction: Lymphocytes were separated from healthy human fetal cord blood by Ficoll lymphocyte separation medium, and CB DNA was extracted by traditional phenol / chloroform method;

[0034] (2) CB DNA transmethylation: 10×NEB buffer 2μl, 1.6mM SAM 2μl, SssI Methylase 4U, CB DNA 1μg, total volume 20μl, positive control DNA was prepared according to the ...

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Abstract

The invention relates to a methylation quantitative test method of APC gene in the DNA of human blood plasma. The methylation quantitative test method obtains venous blood and the blood plasma, prepares a DNA standard curve sample of the blood plasma, extracts the DNA of the blood plasma to be detected, the DNA of a standard curve blood plasma sample and the DNA of healthy human blood plasma, realizes chemical modification of the DNA of the blood plasma to be detected, the standard curve blood plasma sample and the healthy human blood plasma, designs particularity primers and Taqman fluorescent probes according to 707 CpG locus of a 1A sequence in a human APC gene sub-promoter, builds a standard curve according to the augmentation result of the DNA of the standard blood plasma after the augmentation, and implements methylation quantitative result analysis of the sample to be detected. The methylation quantitative test method of the APC gene solves the defects that the blood plasma has less DNA content, high loss rate, DNA degradation and carcinogenic pollutant, etc. The methylation quantitative test method of the APC gene utilizes a primer pair and a Taqman fluorescent probe that aim at the 1A sequence in the APC gene sub-promoter, realizes the detection aiming at the 707 CpG locus in the APC gene sub-promoter with high methylation developing rate, implements quantitative analysis, and can be used in the aspects of cure effect observing as well as prognosis and relapse monitoring of tumor patients.

Description

technical field [0001] The invention relates to a method for detecting biological macromolecules, in particular to a method for quantitatively detecting methylation of APC gene in human plasma DNA. Background technique [0002] Human plasma DNA refers to DNA (cell free DNA), also known as circulating DNA, which is released into peripheral blood by somatic cells under normal circumstances, at a relatively constant level and maintains a balance. In recent years, with the in-depth study of epigenetics and tumorigenesis mechanisms, there is evidence that some tumor suppressor gene promoters undergo methylation changes in the early stages of tumorigenesis, and this change is always accompanied by the entire process of tumor development. In the process of tumor formation, a large number of tumor cells are produced, and a large number of tumor cells die at the same time. The release of tumor-derived nucleic acids into peripheral blood leads to an increase in the total amount of pla...

Claims

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Application Information

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IPC IPC(8): G01N21/00C12Q1/68
Inventor 潘世扬谢而付高丽
Owner 潘世扬
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