MethylHDA: One-pot helicase-dependent amplification with concurrent DNMT1-mediated maintenance of DNA methylation

DNA methylation is an important epigenetic modification involved in gene regulation, genome stability, development, and disease. Analysis of low-abundance methylated DNA often requires DNA amplification. Conventional amplification methods, however, do not preserve methylation marks on newly synthesized DNA, potentially compromising downstream methylation analysis. Recently, DNA methyltransferase 1 (DNMT1) was coupled with phi29-mediated whole-genome amplification to preserve DNA methylation for downstream bisulfite sequencing. Targeted amplification of short, predefined genomic loci requires a different amplification strategy. Here, we developed MethylHDA, a one-pot isothermal workflow that integrates primer-directed helicase-dependent amplification (HDA) with DNMT1-mediated maintenance of DNA methylation. Because standard thermophilic HDA is performed at approximately 65 {degrees}C, a temperature incompatible with sustained DNMT1 activity, we established a unified reaction buffer and operating temperature of 42 {degrees}C that supported both HDA and DNMT1. Under these conditions, HDA produced an approximately five-cycle reduction in qPCR threshold cycle (Ct) relative to the unamplified control. Methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR) demonstrated that inclusion of DNMT1 increased retention of methylation-dependent restriction protection and that the methylation signal was proportional to the methylated fraction of the input templates. These results demonstrate the feasibility of coupling targeted isothermal DNA amplification with concurrent maintenance of DNA methylation in a single reaction. This approach provides a foundation for methylation-preserving preamplification of short, locus-specific targets for downstream MSRE-qPCR and other targeted methylation assays.