Accurate Isothermal Amplification Reaction Rate Determination Using High-Frequency Sampling

BackgroundNucleic acids quantification by amplification is currently done primarily by real-time amplification for relative quantification, or by statistical inference from replicated endpoint assays for absolute quantification. The polymerase chain reaction (PCR) has been the dominant amplification technology, although alternative isothermal technologies have been described. Theoretical analysis of amplification kinetics and of amplification data interpretation have almost exclusively considered the PCR.\n\nResultsReal-time measurements of isothermal amplification reactions can be made continuously, in contrast to the discrete per-cycle measurements of real-time PCR. Isothermal ramified rolling circle amplification (RAM) reactions were measured at frequent intervals, and amplification data subsets were fitted to an exponential amplification model. Signal-change-over-time slopes and time-zero signal intercepts were derived from the chosen subset data. Slope measurements were sufficient to determine a reaction rate (the isothermal equivalent of PCR efficiency) for each reaction. Analysis of slope and intercept together suggest that amplification reactions that were initiated from a single target molecule can be distinguished from reactions that that were initiated from greater than one target molecule.\n\nConclusionsThe constant reaction environment of isothermal nucleic acid amplification allows continuous monitoring of reaction rate. Functional regions of interest in real-time data can be determined directly from the data. Accurate per-reaction efficiency can be readily measured. Improved estimation of low target copy number should improve quantification efficiency.