Synthesis of universal unmethylated control DNA by nested whole genome amplification with ϕ29 DNA polymerase

Optimization of highly sensitive methods to detect methylation of CpG islands in gene promoter regions requires adequate methylated and unmethylated control DNA. Whereas universal methylated control DNA is available, universal unmethylated control (UUC) DNA has not been made because demethylase is not available to remove methyl groups from all methylated cytosines. On the basis that DNA synthesized by DNA polymerase does not contain methylated cytosines, we developed a method to create UUC DNA by nested whole genome amplification (WGA) with ϕ29 DNA polymerase. Contamination of the template genomic DNA in UUC was only 3.1 × 10−7, below the detection limit of sensitive methods used for methylation studies such as methylation-specific PCR. Assessment of microsatellite markers demonstrated that even nested ϕ29 WGA achieves highly accurate and homogeneous amplification with very low amounts of genomic DNA as an initial template. The UUC DNA created by nested ϕ29 WGA is practically very useful for methylation analysis.