MethylAmp One-step isothermal amplification with preservation of DNA methylation patterns

DNA methylation is a critical epigenetic modification that regulates gene expression, maintains genome stability, and influences cellular function during development and disease. Accurate analysis of DNA methylation often requires amplification to generate sufficient material, yet preserving the original epigenetic information during this process is challenging because standard amplification methods can disrupt methylation patterns. To address this, we developed a one-pot strategy that combines helicase-dependent amplification (HDA) with DNA methyltransferase 1 (DNMT1)-mediated methylation, enabling simultaneous DNA amplification and preservation of native methylation marks. A key challenge is that HDA is optimized at 65 {degrees}C, whereas DNMT1 is unstable at elevated temperatures. We overcame this by establishing a unified buffer and isothermal reaction at 42 {degrees}C that supports both enzymatic activities. Under these conditions, HDA achieved robust amplification ([~]5 Ct), while DNMT1 faithfully methylated the newly synthesized DNA, as confirmed by methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR), with methylation levels proportional to the input template. This one-pot workflow demonstrates the feasibility of concurrent amplification and methylation, providing a foundation for scalable, accurate, and methylation-preserving DNA analyses for epigenetic and clinical applications. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/709983v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@fb8d3aorg.highwire.dtl.DTLVardef@f50c94org.highwire.dtl.DTLVardef@cda7aorg.highwire.dtl.DTLVardef@1db82b0_HPS_FORMAT_FIGEXP M_FIG C_FIG