The detection of bacteria, viruses, and genetic mutations rely upon the use of polymerase chain reaction (PCR), whereby nucleic acids are amplified through thermal cycling. But what if you could do PCR without thermal cycling? In a recent paper published in Nature Communications, the researchers engineered an enzyme that can efficiently unwind DNA for PCR, bypassing the need for thermocyclers.
Their method, called SHARP for SSB-Helicase Assisted Rapid PCR, uses the PcrA M6 helicase enzyme they engineered and single-stranded DNA binding protein (SSB), along with standard PCR reagents. In the study, SHARP was used to generate functional DNA and DNA fragments and plasmids, as well as to validate CRISPR-Cas9 editing at endogenous genomic sites.
While various isothermal methods have been previously available, such as loop-mediated isothermal amplification (LAMP), these methods have limitations compared to traditional PCR when it comes to their versatility and the length of DNA that can be amplified. In contrast to these isothermal methods, SHARP can generate amplicons that are up to 6,000 base pairs long.
In addition to generating long amplicons, SHARP performed equally well or better than PCR in terms of amplification time (usually 5–30 min for SHARP versus 45–60 min for 40 cycles of PCR), detection limit, specificity, and appearance as a single band (rather than multiple bands) on a gel. SHARP and PCR can be used in similar ways, including real-time product monitoring with an intercalating dye and downstream applications (purification, sequencing, and cloning). Finally, unlike other isothermal methods, reagents and primers optimized for PCR can be used with SHARP, making the method easy to adopt into clinical and life science labs.