Multiple PCR primer system and detection method for detecting lung cancer circulating free DNA (cfDNA) methylation as well as application
A technology of methylation and plasma, applied in the field of multiple PCR primer system, can solve the problems of low sensitivity and false negative of radiation, sputum exfoliated cell inspection
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Embodiment 1
[0196] Example 1, multiplex PCR primer system for detection of plasma free DNA methylation in lung cancer
[0197] In this embodiment, the primers are designed for the target region of 120 bases upstream and downstream of the lung cancer-specific methylation site. Since the longest length of plasma free DNA generally does not exceed about 200 bp, it is necessary to limit the amplification length of the primers to within 120 bases.
[0198] Table 1 shows the overview of multiplex PCR primers designed in this example for the detection of methylation of plasma cell-free DNA in lung cancer.
[0199] Table 1 Overview of multiplex PCR primer design for detection of plasma cell-free DNA methylation in lung cancer
[0200]
Embodiment 2
[0201] Example 2. Using the multiplex PCR primer system designed by the present invention to detect the methylation level of plasma free DNA in lung cancer
[0202] S1. Extraction of cell-free DNA from lung cancer plasma
[0203] Use the QIAamp Circulating Nucleic Acid Kit (Qiagen 55114) to extract lung cancer plasma cell-free DNA according to the kit instructions.
[0204] S2. Plasma free DNA bisulfite conversion and purification
[0205] Use the EZ DNA Methylation-Gold Kit (zymo Research D5005s) to convert 10 ng of plasma free DNA according to the kit instructions. The specific implementation steps are as follows:
[0206] 1) Prepare the CT Conversion Reagent conversion solution according to the instructions, and prepare it for immediate use;
[0207] 2) Take 20 μL of unconverted DNA sample (sample volume is less than 20 μL, add water to make up), add 130 μL of CTConversion Reagent transformation solution, vortex to mix well, and briefly centrifuge to collect the reaction ...
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