Detection of CpG methylation based on the change in amplification efficiency of strand-displacement DNA polymerase by CpG methylation†

Abstract

A method for detecting CpG methylation is required in clinical settings because CpG methylation is associated with various diseases. CpG methylation leads to structural changes in single-stranded DNA and also changes the stability of double-stranded DNA. We hypothesized that the amplification efficiency of DNA polymerase, with its strand displacement ability, might be altered by CpG methylation. We chose loop-mediated isothermal amplification (LAMP), which uses strand displacement DNA synthesis, for its validation. The LAMP products from the synthetic DNA of the upstream region of the dopamine receptor D2 (DRD2) and the androgen receptor (AR) promoter region were detected by turbidity and fluorescence intensity measurements. The methylated synthetic DNA was amplified more slowly than the unmethylated synthetic DNA. The LAMP products from the human genomic DNA were detected by fluorescence intensity measurement and electrophoresis. The highly methylated genomic DNA was amplified slower than the less methylated genomic DNA in the AR promoter region. CpG methylation detection through differences in the amplification efficiency of LAMP reaction may be used for a rapid and easy detection method of CpG methylation.