Abstract
BACKGROUND: Over the past 40 years, molecular diagnostic methods have evolved from multi-step, time-consuming protocols towards either rapid targeted tests or expansive, massively parallel testing. AIMS: Here we consider the speed limits of targeted molecular diagnostics, considering the three sequential required steps: nucleic acid preparation, amplification, and analysis. MATERIALS & METHODS: Instead of the bind/wash/elute steps commonly used for nucleic acid extraction, simple alkaline lysis of whole blood results in a suspension ready for PCR in seconds that can be added directly to an appropriately buffered PCR master mix. For amplification, the time requirements of PCR are typically limited by the temperature cycling instrumentation and not by biochemistry. RESULTS & DISCUSSION: By lowering sample volumes, increasing the surface area to volume ratio, decreasing the thickness of the sample container, decreasing the amplicon size, and inducing rapid temperature changes by a myriad of innovative means, 30 cycles of PCR can easily be completed in less than 5 min. By increasing primer and polymerase concentrations in synchrony with even faster cycling (< 2 s cycles), "extreme PCR" has amplified a 60 bp human genomic target in < 15 s (35 cycles) with high yield and specificity. For analysis, cumbersome, contamination-prone gel analysis can be replaced by melting curve analysis. Although melting curve analysis usually takes up to an hour on commercial instrumentation, precise temperature control can enable single base genotyping in 1-4 s. CONCLUSION: These advances demonstrate the feasibility of sample-to-answer molecular diagnostics in seconds.