科研之友
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摘要
The hypermethylated tumor suppressor gene promoters are widely recognized as promising markers. for cancer screening and ideal targets for cancer therapy, however, a major obstacle in their clinical study is highly sensitive screening. To address this limitation, we developed a novel bisulfite-microfluidic temperature gradient capillary electrophoresis (bisulfite-mu TGCE) platform for gene methylation analysis by combining bisulfite treatment and slantwise radiative heating system-based mu TGCE. Bisulfite-treated genomic DNA (gDNA) was amplified with universal primers for both methylated and unmethylated sequences, and introduced into glass microfluidic chip to perform electrophorectic separation under a continuous temperature gradient based on the formation of heteroduplexes. Eight CDKN2A promoter model fragments with different number and location of methylation sites were prepared and successfully analyzed according to their electrophoretic peak patterns, with high stability, picoliter-scale sample consumption, and significantly increased detection speed. The bisulfite-mu TGCE could detect methylated gDNA with a detection limit of 7.5 pg, and could distinguish as low as 0.1% methylation level in CDKN2A in an unmethylated background. Detection of seven colorectal cancer (CRC) cell lines with known and unknown methylation statuses of CDKN2A promoter and 20 tumor tissues derived from CRC patients demonstrated the capability of detecting hypermethylation in real-world samples. The wider adaptation of this platform was further supported by the detection of the CDKN2A and MLH1 promoters' methylation statuses in combination. This highly sensitive, fast, and low-consumption platform for methylation detection shows great potential for future clinical applications.
- 出版日期2013-4-15
- 单位中国医科大学
