Reverse transcriptase kinetics for one-step RT-PCR

Abstract

Understanding reverse transcriptase (RT) activity is critical for designing fast one-step RT-PCRs. We report a stopped-flow assay that monitors SYBR Green I fluorescence to investigate RT activity in PCR conditions. We studied the influence of PCR conditions on RT activity and assessed the accuracy of cDNA synthesis predictions for one-step RT-PCR. Nucleotide incorporation increased from 26 to 89 s^-1 between 1.5 and 6 mM MgCl₂ but was largely unaffected by changes in KCl. Conversely, increasing KCl from 15 to 75 mM increased apparent rate constants for RT-oligonucleotide binding (0.010-0.026 nM^-1 s^-1) and unbinding (0.2-1.5 s^-1). All rate constants increased between 22 and 42 °C. When evaluated by PCR quantification cycle, cDNA predictions differed from experiments using RNase H+ RT (average 1.7 cycles) and RNase H- (average 4.5 cycles). Decreasing H+ RT concentrations 10 to 10⁴-fold from manufacturer recommendations improved cDNA predictions (average 0.8 cycles) and increased RT-PCR assay efficiency. RT activity assays and models can be used to aid assay design and improve the speed of RT-PCRs. RT type and concentration must be selected to promote rapid cDNA synthesis but minimize nonspecific amplification. We demonstrate 2-min one-step RT-PCR of a Zika virus target using reduced RT concentrations and extreme PCR.

Keywords: Extreme RT-PCR; One-step RT-PCR; Reverse transcriptase assay; Steady-state kinetics; Transient-state kinetics; cDNA predictions.