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...

Thermal denaturations of the type 1 DNA polymerases from Thermus aquaticus (Taq polymerase) and Escherichia coli (Pol 1) have been examined using differential scanning calorimetry and CD spectroscopy. The full-length proteins are single-polypeptide chains comprising a polymerase domain, a proofreading domain (inactive in Taq) and a 5' nuclease domain. Removal of the 5' nuclease domains produces...

Oligonucleotide-directed mutagenesis is an important method for modifying specific bases of a DNA sequence (8). Current approaches include combining inverse polymerase chain reaction (PCR) with long PCR to introduce mutations directly into an intact plasmid template (1,5,10). However, the use of Taq DNA polymerase in these protocols results in a high rate of base mis-incorporation (3,7). Moreov...

To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division Proteobacteria as well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR artif...

DNA polymerases contain active sites that are structurally superimposable and conserved in amino acid sequence. To probe the biochemical and structure-function relationship of DNA polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (Motif A; amino acids 605-6...

The polymerase chain reaction (PCR) has revolutionized the molecular biology industry, with profound implications for medical, forensic and research sciences. Prerequisites for this technology to work include the presence of deoxynucleotriphosphates (dNTPs), a relevant buffering system, magnesium chloride, primers and template DNA and finally, the DNA polymerase. Taq polymerase, a thermostable ...