Fluorescence polarization-based method with bisulfite conversion-specific one-label extension for quantification of single CpG dinucleotide methylation.
To quantify the methylation at individual CpG dinucleotide sites in large biological or clinical samples, we developed a bisulfite conversion-specific one-label extension (BS-OLE) method using visualization by fluorescence polarization (FP) measurement of methylation at single CpG sites in small amounts of genomic DNA. Genomic DNA was treated with sodium bisulfite to convert unmethylated cytosine to uracil leaving 5-methylcytosine unaltered, and BS-PCR was used to generate DNA template containing target CpG sites. BS-OLE uses a BS-primer hybridized immediately upstream of the target CpG site being examined and then fluorescent dCTP or dUTP is incorporated into the methylated (CpG) or unmethylated (TpG) form of the target site through single-nucleotide chain extension, yielding an FP ratio between the fluorescent dCTP- and dUTP-incorporated products as a measure of methylation. This provides stable estimates of the methylation level of human genomic DNA and of a 250-bp plasmid DNA segment containing a single TCGA TaqI cleavage site, in accordance with the results of a combined bisulfite restriction analysis method. We used BS-OLE to measure dose-dependent DNA hypomethylation in human embryonic kidney 293T cells treated with the DNA methyltransferase inhibitor 5-aza-dC. BS-OLE is well suited to high-throughput multi-sample applications in biological and medical studies.
Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, No. 1023 Shatai Road, Baiyun District, Guangzhou City, Guangdong Province 510515, China.|Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, No. 1023 Shatai Road, Baiyun District, Guangzhou City, Guangdong Province 510515, China.
Shufen
S
Li
Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, No. 1023 Shatai Road, Baiyun District, Guangzhou City, Guangdong Province 510515, China.|Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, No. 1023 Shatai Road, Baiyun District, Guangzhou City, Guangdong Province 510515, China.
Zhongju
Z
Wang
Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, No. 1023 Shatai Road, Baiyun District, Guangzhou City, Guangdong Province 510515, China.|Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, No. 1023 Shatai Road, Baiyun District, Guangzhou City, Guangdong Province 510515, China.
Lin
L
Zhou
Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, No. 1023 Shatai Road, Baiyun District, Guangzhou City, Guangdong Province 510515, China.|Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, No. 1023 Shatai Road, Baiyun District, Guangzhou City, Guangdong Province 510515, China.
Fu
F
Luo
Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, No. 1023 Shatai Road, Baiyun District, Guangzhou City, Guangdong Province 510515, China.|Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, No. 1023 Shatai Road, Baiyun District, Guangzhou City, Guangdong Province 510515, China.
Cunyou
C
Zhao
D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D005453:Fluorescence; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*
BS-OLE; DNA methylation; bisulfite conversion; conversion au bisulfite; extension d’une seule base; fluorescent dCTP/dUTP; méthylation de l’ADN; ratio dCTP/dUTP fluorescents; single-nucleotide extension