The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6)) < < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.

This structure represents the highest resolution view of a Pol I enzyme obtained to date. Comparison of the three Pol I structures reveals no compelling evidence for many of the specific interactions that have been proposed to induce thermostability, but suggests that thermostability arises from innumerable small changes distributed throughout the protein structure. The polymerase domain is highly conserved in all three proteins. The N-terminal domains are highly divergent in sequence, but retain a common fold. When present, the 3'-5' proofreading exonuclease activity is associated with this domain. Its absence is associated with changes in catalytic residues that coordinate the divalent ions required for activity and in loops connecting homologous secondary structural elements. In BF, these changes result in a blockage of the DNA-binding cleft.

The QuikChange Multi Site-Directed Mutagenesis Kit is a simple and efficient method for introducing point mutations at up to five sites simultaneously in plasmid DNA templates. Here we used the QuikChange Multi kit with degenerate (one codon) primers to introduce all possible amino acids at selected sites in the lacZ gene. In reactions employing two or three degenerate primers, diverse libraries (10(4)-10(5) mutants/reaction) are created consisting of random combinations of mutations at two or three different sites. This method provides a one-day procedure for performing site-directed saturation mutagenesis and, when coupled with a suitable screening assay, should greatly facilitate the process of evaluating alternative amino acid chain substitutions at key residues and evolving protein function.

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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.

We discovered a thermostable enzyme from the archaeon Pyrococcus furiosus (Pfu), which increases yields of PCR product amplified with Pfu DNA polymerase. A high molecular mass (>250 kDa) complex with PCR-enhancing activity was purified from Pfu extracts. The complex is a multimer of two discrete proteins, P45 and P50, with significant similarity to bacterial dCTP deaminase͞dUTPase and DNA flavoprotein, respectively. When tested in PCR, only recombinant P45 exhibited enhancing activity. P45 was shown to function as a dUTPase, converting dUTP to dUMP and inorganic pyrophosphate. Pfu dUTPase improves the yield of products amplified with Pfu DNA polymerase by preventing dUTP incorporation and subsequent inhibition of the polymerase by dU-containing DNA. dUTP was found to accumulate during PCR through dCTP deamination and to limit the efficiency of PCRs carried out with archaeal DNA polymerases. In the absence of dUTP inhibition, the combination of cloned Pfu DNA polymerase and Pfu dUTPase (PfuTurbo DNA polymerase) can amplify longer targets in higher yield than Taq DNA polymerase. In vivo, archaeal dUTPases may play an essential role in preventing dUTP incorporation and inhibition of DNA synthesis by family B DNA polymerases.T he use of high-fidelity DNA polymerases in the polymerase chain reaction (PCR) is important for minimizing amplification errors in products that will be cloned, sequenced, and expressed. Several archaeal DNA polymerases with 3Ј-to 5Ј-exonucleasedependent proofreading activity have been commercialized for high-fidelity PCR amplification. Of the enzymes characterized to date, Pyrococcus furiosus (Pfu) DNA polymerase exhibits the highest fidelity, with an average error rate estimated to be 2-to 60-fold lower than other proofreading enzymes and 6-to 100-fold lower than Taq DNA polymerase (1-4), which lacks proofreading activity.Although inherent high fidelity and other unique biochemical properties of Pfu DNA polymerase have been exploited in numerous molecular biology procedures, including blunt-end cloning (5), amplification of GC-rich targets (6), mutation detection (7), ligation-independent cloning (8), site-directed mutagenesis (9), genotyping (10), and trinucleotide repeat analyses (11), it has been reported that proofreading DNA polymerases tend to perform less reliably than Taq DNA polymerase (4, 12). PCRs conducted with Taq are typically more efficient and require less optimization than reactions carried out with proofreading DNA polymerases. Lower product yields with Pfu DNA polymerase have been attributed to the enzyme's relatively slow polymerization rate (550 vs. 2,800 nucleotides per min for Taq) and interference from its associated 3Ј-to 5Ј-exonuclease activity (13).Improvements in the performance of Pfu DNA polymerase have been achieved by increasing PCR extension times (2 min͞kb of target; Stratagene Pfu DNA polymerase manual), incorporating additives such as DMSO (6,14), and mixing Pfu with Taq DNA polymerase (4). Although DNA polymerase mixtures synthesize higher yields of produ...

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We have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy-and K light-chain variable and constant region domains, were inserted into modified bacteriophage A expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2% in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. We estimate that this human immunoexpression library contains 20,000 clones with high aftmity and specificity to our chosen antigen.