Quantitative analysis of DNA methylation using sequence-specific, real-time loop-mediated isothermal amplification

Abstract

A number of genetic diseases and in the majority of tumor diseases have been reported to be associated with anomalous methylation levels of DNA. Accurate determine of the methylation level is an essential prerequisite for epigenetic diagnostics. Although some studies have used probe with a quencher enable real-time monitor of loop-mediated isothermal amplification (LAMP), most of them lack systematic investigations of design principle and applicable ambient when applied in LAMP based methylation detection. Herein, a sequence-specific, real-time loop-mediated isothermal amplification was developed for the quantitative analysis of DNA methylation. An oligonucleotide dimer was designed to implement three functions at the same time, including primer, sense probe and signal probe (PSS probe). Systematic investigations were performed to optimize the performance of PSS probe. After that, our method can accurately determine as low as 0.1% synthetic methylated DNA in the presence of numerous unmethylated DNA. Moreover, a higher methylated level of kazrin periplakin interacting protein (KAZN) gene was detected in colorectal cancer cells with a stable housekeeping gene β-actin used as an endogenous control. The developed PSS probe for LAMP provide an option to analyze DNA methylation levels in resource-limited environments, and hold great potential for the diagnosis of methylation-related diseases.