An improved bisulfite genomic sequencing for DNA methylation marker scanning

Objective To develop a simplified bisulfite genomie sequencing(BGS)method for DNA methylation marker scanning.Methods According to modified BGS protocol,the desalt DNA treated with bisulfite were directly used for bisulfite-PCR(BSP)without alkali treatmenL Complement of the bisulfite modification Wag accomplished by a prolonged pre-denaturation stage.After BSP,a second round PCR was performed with a pair of GC tagged primers to adjust the GC content of the amplieon for direct sequencing.To assess this improved protocol,promotor methylation of TNF-α gene in 3T3-L1 cell and androgen receptor(AR)gene in Hela cell was investigated.The real time BSP for Alu was also used to compare the sensitivity of the modified assay with traditional assay.Results Both the hypermethylated TNF-α promotor and hypomethylated AR promotor were successfully sequenced by improved BGS method,and the results were consistent with that of the traditional assay.The conversion rate reached 100%,while the conversion specificity was higher than 93.75%.The sensitivity of improved BGS method inereaged significantly(t=2.978 2,P＜0.05)and showed good reproducibility.Condusion The improved BGS method is simple and sensitive,facilitating more ambitious genomic methyhtion mapping studies. Key words: Bisulfite genomie sequencing; DNA methylation; Bisulfite; Polymerase chain reaction