mPCR/SERS assay for hMLH1 promoter methylation identification

Abstract

Gene-specific DNA methylation is associated with the progression of various cancers. Thus, accurate identification of this epigenetic alteration is of great interest. In this study, we particularly focused on the use of surface-enhanced Raman scattering (SERS) for detecting methylation in the promoter region of the hMLH1 gene found in cancer cells. A promoter segment from two different colorectal cancer cell lines, SW48 and SW480 identified as hypermethylated and unmethylated respectively, was amplified by methylation-specific PCR (mPCR). This produced two different types of amplicons with specific primers where the forward primer was labelled with completely different 5′ overhang sequences, and the reverse primer was labelled with biotin. This allowed them to bind specifically with either a methylation-specific nanotag (mSERS) or an unmethylation-specific nanotag (umSERS), followed by enrichment by streptavidin magnetic beads (SMB). This resulted in the formation of a nanotag-amplicon-SMB complex that could be easily isolated from the unbound molecules. Finally, SERS analysis of the complex produced specific spectral profiles related to mSERS and umSERS nanotags, indicating the methylation status of the promoters. The mPCR/SERS assay demonstrated a limit of detection (LOD) of 3.93% for sensing DNA methylation with high specificity. Thus we believe the proposed assay can find more broad applications for methylation analysis in any DNA samples.