Abstract
DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.
Highlights
On the basis of their amino acid sequence and structural analysis, DNA polymerases have been classified into seven families, A, B, C, D, E, X, and Y (Delarue et al, 1990; Braithwaite and Ito, 1993; Joyce and Steitz, 1994; Cann et al, 1998; Ishino et al, 1998; Ohmori et al, 2001; Lipps et al, 2003)
We found valuable biochemical properties in P. abyssi (Pab)-polD compared to the conventional Thermus aquaticus (Taq), which makes the enzyme ideally suited for cutting-edge polymerase chain reaction (PCR)-applications
The optimized buffer for PCR with Pab-polD was obtained by varying different components of the standard Pab-polB reaction buffer
Summary
On the basis of their amino acid sequence and structural analysis, DNA polymerases have been classified into seven families, A, B, C, D, E, X, and Y (Delarue et al, 1990; Braithwaite and Ito, 1993; Joyce and Steitz, 1994; Cann et al, 1998; Ishino et al, 1998; Ohmori et al, 2001; Lipps et al, 2003). Beginning with the discovery and characterization of DNA polymerase I (Family A) from Thermus aquaticus (Taq) (Chien et al, 1976), a variety of thermostable DNA polymerases have been isolated and identified from prokaryotic organisms. Besides their crucial biological functions, thermostable DNA polymerases have proven to be technically and economically important enzymes. They are versatile tools used in DNA technologies such as cycle sequencing and polymerase chain reaction (PCR) (Pavlov et al, 2004).
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