title: A simple method for high-throughput quantification of genome-wide DNA methylation by fluorescence polarization.
abstract: To rapidly determine DNA methylation levels from a large number of biological or clinical samples, we have developed an accurate and sensitive method for high-throughput quantification of global methylation of 5'-Cm ( 5) CGG-3' sites in the genome, visualized by fluorescence polarization (FP) based measurement of DNA methylation (FPDM). In FPDM, the methyl-sensitive HpaII and methyl-insensitive MspI restriction enzymes were employed to achieve DNA cleavage, followed by incorporation of fluorescent dCMP into the enzyme-cleavage products through polymerase chain extension, yielding an FP-ratio between the HpaII- and MspI-restricted preparations as a measure of methylation. FPDM provided stable estimates of methylation level of submicrograms of lambda or human DNA, and of a 255-bp DNA segment containing a single HpaII/MspI restriction site in accord with, and more accurate than, determination by gel electrophoresis. FPDM was also applied to measure dose-dependent DNA hypomethylation in human embryonic kidney 293T cells treated with the DNA-methyltransferase inhibitor 5-aza-dC.
authors.affiliation: Division of Life Science and Applied Genomics Center, The Hong Kong University of Science and Technology, Hong Kong, China. zhaocunyou@gmail.com
authors.forename: Cunyou
authors.initials: C
authors.lastname: Zhao
authors.forename: Hong
authors.initials: H
authors.lastname: Xue
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D010582:Bacteriophage lambda / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D018899:CpG Islands; D019175:DNA Methylation; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D000077209:Decitabine; D019003:Deoxyribonuclease HpaII / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D015894:Genome, Human; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D004279:DNA, Viral; C495710:GABRB2 protein, human; D011963:Receptors, GABA-A; D000077209:Decitabine; D019003:Deoxyribonuclease HpaII; D001374:Azacitidine