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In an effort to increase the thermostability of Moloney Murine Leukemia Virus reverse transcriptase (MMLV RT), we screened random and site-saturation libraries for variants that show increased resistance to thermal inactivation. We discovered five mutations E69K, E302R, W313F, L435G and N454K that collectively increase the half-life of MMLV RT at 55°C from less than 5 min to ∼30 min in the presence of template-primer. In addition, these mutations alter the thermal profile by increasing specific activity of the pentuple mutant (M5) over a broad range of cDNA synthesis temperatures (25–70°C). We further show that M5 generates higher cDNA yields and exhibits better RT–PCR performance compared to wild-type RT when used at high temperature to amplify RNA targets containing secondary structure. Finally, we demonstrate that M5 exhibits tighter binding (lower Km) to template-primer, which likely protects against heat inactivation.

Regulation of the p49-p58 primase complex during primer synthesis and the interaction of the primase subunits with DNA were examined. After primase synthesizes a primer that DNA polymerase alpha (pol alpha) can readily elongate, further primase activity is negatively regulated. This occurs within both the context of the four-subunit pol alpha-primase complex and in the p49-p58 primase complex, indicating that the newly generated primer-template species need not interact with pol alpha to regulate further primase activity. Photo-cross-linking of single-stranded DNA-primase complexes revealed that whereas the isolated p49 and p58 subunits both reacted with DNA upon photolysis, only the p58 subunit reacted with the DNA when photolysis was performed using the p49-p58 primase complex. After primer synthesis by the complex, p58 was again the only subunit that reacted with the DNA. These results suggest a model for regulation of primer synthesis in which the newly synthesized primer-template species binds to p58 and regulates further primer synthesis. Additionally, the ability of p58 to interact with primer-template species suggests that p58 mediates the transfer of primers from the primase active site to pol alpha.

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Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.

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