The bisulfite genomic sequencing (BGS) proto- col has gained worldwide
popularity as the method of choice for analyzing DNA methylation. It is this
popular because it is a powerful protocol and it may be coupled with many other
applications. However, users often run into a slew of problems, including
incomplete conversion, overly degraded DNA, sub-optimal PCR amplifications,
false positives, uninformative results, or altogether failed experiments. We
pinpoint the reasons why these problems arise and carefully explain the
critical steps toward accomplishing a successful experiment step-by-step. This
protocol has worked successfully (>99.9% conver- sion) on as little as 100
ng of DNA derived from nearly 10-year-old DNA samples extracted from whole
blood stored at −80°C and resulted in enough converted DNA for more than 50 PCR
reactions. The aim of this article is to make learning and usage of BGS easier,
more efficient and standardized for all users.