Abstract
Isothermal PCR can be performed by imposing a static temperature gradient that continuously circulates reagents through denaturing, annealing, and extension conditions inside a PCR tube. But despite early promise, these systems have yet to demonstrate performance and repeatability sufficient for adoption in validated laboratory tests because the rate-limiting extension step is inherently short and cannot be increased independently of the other stages in a temperature cycle. Here, a discovery that enables isothermal PCR to be achieved with statistically robust repeatability that meets or exceeds diagnostic assay requirements (false positive/negative rate <8% at 95% confidence) by manipulating the interplay between the DNA replication biochemistry (via the amplicon GC content) and the microscale circulatory flow inside a PCR tube is reported. Surprisingly, optimal performance depends on selecting primer sequences that replicate high GC content amplicons, contradicting established PCR primer design rules. This innovative thermocycling approach accelerates PCR to speeds rivaling ultra-fast instruments, enabling rapid, repeatable isothermal DNA analysis across a range of targets relevant to diagnostics and pathogen detection.