Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance.

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.

Engineered split in Pfu DNA polymerase fingers domain improves incorporation of nucleotide gamma-phosphate derivative.

Using compartmentalized self-replication (CSR), we evolved a version of Pyrococcus furiosus (Pfu) DNA polymerase that tolerates modification of the γ-phosphate of an incoming nucleotide. A Q484R mutation in α-helix P of the fingers domain, coupled with an unintended translational termination-reinitiation (split) near the finger tip, dramatically improve incorporation of a bulky γ-phosphate-O-linker-dabcyl substituent. Whether synthesized by coupled translation from a bicistronic (-1 frameshift) clone, or reconstituted from separately expressed and purified fragments, split Pfu mutant behaves identically to wild-type DNA polymerase with respect to chromatographic behavior, steady-state kinetic parameters (for dCTP), and PCR performance. Although naturally-occurring splits have been identified previously in the finger tip region of T4 gp43 variants, this is the first time a split (in combination with a point mutation) has been shown to broaden substrate utilization. Moreover, this latest example of a split hyperthermophilic archaeal DNA polymerase further illustrates the modular nature of the Family B DNA polymerase structure.

Mutant of Moloney murine leukemia virus reverse transcriptase exhibits higher resistance to common RT-qPCR inhibitors.

Inhibitor resistance of several commercial Moloney murine leukemia virus reverse transcriptase (MMLV RT) enzymes was investigated. IC(50) values were determined for potential RNA contaminants, including guanidine thiocyanate, ethanol, formamide, ethylenediaminetetraacetic acid (EDTA), and plant-related acidic polysaccharides. Sensitivity (as judged by MMLV RT IC(50) values) was directly correlated to the outcome of "mock" reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays carried out with exogenous inhibitors. MMLV RT enzymes lacking RNase H activity were shown to be more sensitive to RT-qPCR inhibitors. In contrast, a thermal-resistant MMLV RT pentuple mutant (E69K/E302R/W313F/L435G/N454K) showed higher tolerance to these substances than the wild type. Increased resistance was also noted in RT-qPCR comparisons employing crude cell lysates.

Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer.

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 degrees C from less than 5 min to approximately 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 degrees 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 K(m)) to template-primer, which likely protects against heat inactivation.

Escherichia coli DNA polymerase III epsilon subunit increases Moloney murine leukemia virus reverse transcriptase fidelity and accuracy of RT-PCR procedures.

In an effort to improve reverse transcriptase (RT) fidelity, we measured the error rate of Moloney murine leukemia virus (MMLV) RT in the presence of several autonomous and DNA polymerase-associated 3'-5' exonucleases using a lacZ forward mutation assay. A number of 3'-5' exonucleases were found to lower the error rate of MMLV RT, including p53, Escherichia coli DNA polymerase III epsilon subunit, and the proofreading activities associated with T4, varphi29, and E. coli pol I DNA polymerases. The bacterial epsilon subunit increased RNA-dependent DNA synthesis fidelity by approximately threefold and was the only 3'-5' exonuclease tested that did not deleteriously affect RT-PCR yields. Further testing showed that RT-PCR mutant frequencies were reduced significantly by performing cDNA synthesis in the presence of epsilon subunit, followed by PCR with a high-fidelity proofreading DNA polymerase. DNA sequence analysis was used to show that the combination of MMLV RT/epsilon subunit and PfuUltra DNA polymerase produces approximately eightfold fewer errors compared with the commonly used combination of MMLV RT and a Taq-based high-fidelity blend, consistent with predictions based on experimentally determined polymerase error rates.

Amplification efficiency of thermostable DNA polymerases.

The amplification efficiencies of several polymerase chain reaction (PCR) enzymes were compared using real-time quantitative PCR with SYBR Green I detection. Amplification data collected during the exponential phase of PCR are highly reproducible, and PCR enzyme performance comparisons based upon efficiency measurements are considerably more accurate than those based on endpoint analysis. DNA polymerase efficiencies were determined under identical conditions using five different amplicon templates that varied in length or percentage GC content. Pfu- and Taq-based formulations showed similar efficiencies when amplifying shorter targets (<900 bp) with 45 to 56% GC content. However, when amplicon length or GC content was increased, Pfu formulations with dUTPase exhibited significantly higher efficiencies than Taq, Pfu, and other archaeal DNA polymerases. We discuss the implications of these results.

Efficient and high fidelity incorporation of dye-terminators by a novel archaeal DNA polymerase mutant.

We examined the molecular basis of ddNTP selectivity in archaeal family B DNA polymerases by randomly mutagenizing the gene encoding Thermococcus sp. JDF-3 DNA polymerase and screening mutant libraries for improved ddNTP incorporation. We identified two mutations, P410L and A485T, that improved ddNTP uptake, suggesting the contribution of P410 and A485 to ddNTP/dNTP selectivity in archaeal DNA polymerases. The importance of A485 was identified previously in mutagenesis studies employing Pfu (A486) and Vent (A488) DNA polymerases, while the contribution of P410 to ddNTP/dNTP selectivity has not been reported. We demonstrate that a combination of mutations (P410L/A485T) has an additive effect in improving ddNTP incorporation by a total of 250-fold. To assess the usefulness of the JDF-3 P410L/A485T in fluorescent-sequencing applications, we compared the archaeal mutant to Taq F667Y with respect to fidelity and kinetic parameters for DNA and dye-ddNTPs. Although the Taq F667Y and JDF-3 P410L/A485T mutants exhibit similar K(m) and V(max) values for dye-ddNTPs in single-base extension assays, the archaeal mutant exhibits higher fidelity due to a reduced tendency to form certain (ddG:dT, ddT:dC) mispairs. DNA polymerases exhibiting higher insertion fidelity are expected to provide greater accuracy in SNP frequency determinations by single-base extension and in multiplex minisequencing assays.