Direct quantitative bisulfite sequencing using tag-modified primers and internal normalization

Abstract

For the investigation of DNA methylation patterns, bisulfite conversion of the DNA followed by polymerase chain reaction (PCR) amplification and sequencing of the region of interest is the method of choice when information at single CpG site resolution is desired. In this study, a simple method for direct quantitative bisulfite sequencing based on the Sanger method is shown to be usable for the accurate analysis of single CpG sites. This method is based on the usage of tagmodified primers to obtain an internal normalization signal within the PCR product.