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DNA methylations at specific CpG loci have been increasingly utilized as biomarkers for cancer diagnostics.Complex multistep procedures of methylation assay such as DNA extraction,bisulfite conversion can decreased sensitivity and accuracy of quantification.Here we described a highly reproducible,endonuclease-based,quantitative and high throughput CpG methylation assay that does not involve DNA extraction and bisulfite conversion,with an ELISA-like workflow on one 96-well plate.