Bisulfite modification of immobilized DNAs for methylation detection.
We have developed a novel method for detecting DNA methylation status of multiple samples, in which the DNA samples were firstly immobilized on the slide and treated with bisulfite directly on the chip. In this experiment, DNAs of pUC19 plasmid were restricted by the enzymes, and ligated with a linker bearing 5'-terminal acrylamide group at the sticky ends. Using universal acrylamide gel polymerization technique, a large amount of DNAs could be immobilized on the slide. The immobilized DNAs were converted by soaking the chip in bisulfite reaction mixtures for 16 h. The probes for detection of the methylation patterns of CpG sites hybridized with the converted DNAs on the microarray, and non-specifically bound probes were cleaned by electrophoresis. We have optimized the experimental conditions of both bisulfite treatment and electrophoresis to increase sensitivity and specificity. The results were further validated by bisulfite DNA sequencing. The experiments show that the method can simplify the experimental processes and increase the efficiency of the bisulfite treatment. This novel method could be used as a convenient tool to detect the methylation status of the multiple genes for a large amount of samples in the future.
Wan
Yuan
Y
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, PR China.
Wang
Yan
Y
Luo
JunFeng
J
Lu
ZuHong
Z
D000327:Adsorption; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D019175:DNA Methylation; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000737:chemistry*
D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite