Relay-MSRE-qPCR: A sequential multi-buffer staged-dilution workflow for methylation-sensitive restriction enzyme qPCR

Methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR) quantifies locus-specific DNA methylation in native DNA without bisulfite conversion. However, such multi-enzyme workflows are limited by buffer incompatibility, cumulative inhibitor carry-over, and sample loss during purification. We developed Relay-MSRE-qPCR, a sequential staged-dilution workflow in which each heat-inactivated digest is transferred to a fresh tube, diluted ≥5-fold to attenuate inhibitors, and then the buffer for the next enzyme is reconstituted to 1 × concentration. Buffer budgeting kept carry-over compatible with qPCR, with ≤5% (v/v) glycerol included per step. With Eco52I (XmaIII), HhaI, EcoRII, VpaK11BI (AvaII), and HpaII, the relay maintained restriction-site-responsive digestion and the cumulative amount of salts, Mg²⁺, and stabilizers remained within polymerase-tolerable limits; defined unmethylated PCR-product controls showed marked loss of amplifiable template for all five enzymes. Relay and single-enzyme comparator conditions showed consistent delta cycle threshold (ΔCt) behavior, and qPCR products exhibited single-peak melt curves under the tested conditions. Only enzymes recognizing a site within the PCR amplicon produced ΔCt shifts, confirming site-specific responsiveness. A practical protocol is provided with guidance for amplicon placement, enzyme selection, and buffer accounting. Relay-MSRE-qPCR enables reproducible, low-cost targeted CpG methylation analysis when base-resolution sequencing is unnecessary or impractical. Sequential staged dilution preserves enzyme-specific digestion conditions without inter-step purification. Reduces inhibition and enhances reproducibility by budgeting carry-over components within accepted qPCR tolerance ranges. Site-specific ΔCt shifts support reliable targeted CpG methylation analysis.