Quantal PCR: A Calibrator-Free Method for Determining the Unit for Nucleic Acid Quantification

Abstract Quantitative polymerase chain reaction (qPCR) is limited in measuring absolute nucleic acid copy numbers due to the inherent variability of calibrators. Here, we introduce the Quantal PCR (quPCR), a novel method that eliminates the need for calibrators by defining an intrinsic quantal unit derived from the thermodynamic and kinetic properties of the replication system. This approach first determines amplification efficiency at high template concentrations, which is then used as the replication probability to construct quantification cycle (Cq) distribution profiles. These profiles are compared with those from limiting dilution PCR to derive the Cq value for the minimal quantal-replication unit (“quCq”), enabling calculation of the sample copy number. Validation using a dual-target DNA template showed near-identical copy numbers using two distinct target-specific replication systems. Thus, quPCR represents a new method for absolute nucleic acid quantification at the single-molecule level, offering a calibrator-free alternative for absolute quantification. Competing Interest Statement Author X.T. is the inventor on patents related to the methodology described in this paper. This potential conflict of interest has been reviewed and managed by the author’s company. Footnotes The revisions are as follows: 1) In Figure 1, the value 38.24 in the legend is revised to 39.24; 2) In the Supplementary qPCR and linear PCR protocols, the primer concentrations for the R gene and H gene are adjusted; 3) In the Supplementary Figure S4 legend, “Ct” is revised to “Cq”; 4) The legend of Supplementary Table S4 is revised.