Highly Sensitive Detection Of DNA Methylation Based On Loop-mediated Isothermal Amplification (LAMP) Reactions

DNA methylation is often found at cytosines of CpG dinucleotides in genomic DNA by the addition of a methyl group to the C5-position of cytosines, which is the most common a primary epigenetic mechanism for transcriptional regulation of gene during normal development and goes awry in many human diseases, including cancers. The advanced "next-generation sequencing (NGS)" technologies are now enabling the global mapping of DNA methylation at single-base resolution, and providing new insights into the association between the CpG methylation in genomic DNA and cancer occurrence, so DNA methylation has been increasingly utilized as the biomarker for cancer diagnosis. These developments have highlighted the demand for accurate and sensitive detection of CpG methylation in genomic DNA to be routinely used for medical diagnosis with low cost and without requirement of expensive instrumentation. Currently, the PCR-based assays are most widely used for sensitive detection of CpG methylation, however some challenges of the PCR-based assay for practical applications remain to be overcome. Firstly, the PCR-based methods are prone to false positive signal caused by non-specific amplification. Moreover Most of the PCR-based assays require labor-intensive steps or expensive instruments. Some newly developed PCR-free methods generally have low sensitivity, which cannot meet the need for detection of CpG methylation in genomic DNA samples.In order to address the above-mentioned challenges in DNA methylation assay, in this work, we have developed a novel Loop-mediated isothermal amplification (LAMP)-based methylation assay by using methylation-sensitive restriction endonuclease (Hpall) to specifically discriminate between methylated and unmethylated DNA. The DNA Targets are firstly treated with Hpall, where the DNA Targets will be cleaved at specific unmethylated-cytosine residues while remaining the methylated DNA intact. Subsequently, the loop-mediated isothermal amplification (LAMP) can quickly amplify the methylated DNA under isothermal conditions with ultrahigh sensitivity and near zero background. Therefore, the significant advantages of ultrahigh sensitivity and selectivity over conventional methods of methylation detection can be achieved. With the LAMP-based methylation assay, as low as 10 aM methylated DNA Targets can be detected and 0.1% methylated DNA can be determined in the presence of a large excess of unmethylated DNA. Moreover, the proposed assay is performed under isothermal conditions with real-time measurements by using a common fluorescent dye SG without requirement of any DNA label, which can enable simple and cost-effective detection of DNA methylation. The new LAMP-based methylation assay can also be well applied to detection of CpG methylation in genomic DNA. As low as 100 pg genomic DNA can be well determined. Therefore, we believe the proposed assay has great potential for studies of disease diagnosis and biological functions of DNA methylation.