DNA methylation detection based on difference of base content

India-Japan Expert Group Meeting on Biomolecular Electronics & Organic Nanotechnology for Environment Preservation

物理学;生物科学;生态环境科学

Sato, Shinobu^1,2 ; Ohtsuka, Keiichi^1,2 ; Honda, Satoshi^1 ; Sato, Yusuke^1 ; Takenaka, Shigeori^1,2

Department of Applied Chemistry, Kyushu Institute of Technology, Kitakyushu, Japan^1

Research Center for Bio-microsensing Technology, Kyushu Institute of Technology, Kitakyushu, Japan^2

关键词: Cancer markers;    DNA Methylation;    DNA samples;    DNA Sequencing;    Double stranded DNA;    Human genomes;    PCR products;    Two different wavelengths;   

Others  :  https://iopscience.iop.org/article/10.1088/1742-6596/704/1/012015/pdf
DOI  :  10.1088/1742-6596/704/1/012015

来源: IOP

【 摘 要 】

Methylation frequently occurs in cytosines of CpG sites to regulate gene expression. The identification of aberrant methylation of certain genes is important for cancer marker analysis. The aim of this study was to determine the methylation frequency in DNA samples of unknown length and/or concentration. Unmethylated cytosine is known to be converted to thymine following bisulfite treatment and subsequent PCR. For this reason, the AT content in DNA increases with an increasing number of methylation sites. In this study, the fluorescein-carrying bis-acridinyl peptide (FKA) molecule was used for the detection of methylation frequency. FKA contains fluorescein and two acridine moieties, which together allow for the determination of the AT content of double-stranded DNA fragments. Methylated and unmethylated human genomes were subjected to bisulfide treatment and subsequent PCR using primers specific for the CFTR, CDH4, DBC1, and NPY genes. The AT content in the resulting PCR products was estimated by FKA, and AT content estimations were found to be in good agreement with those determined by DNA sequencing. This newly developed method may be useful for determining methylation frequencies of many PCR products by measuring the fluorescence in samples excited at two different wavelengths.