The thermostable properties of the DNA polymerase activity from Thermus aquaticus (Taq) have contributed greatly to the yield, specificity, automation and utility of the polymerase chain reaction method for amplifying DNA (1). We report here the cloning and nucleotide sequence of a DNA polymerase gene from another thermophilic bacterium — Thermus flavus (Tfl). The cloning of the DNA polymerase ...

The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N. equitans family B DNA polymerase (Neq DNA polymerase) produced lambda DNA fragments up to...

optimization of the condition for pcr-directed sequencing of microsatellites poly adenine (a) length polymorphisms is more difficult and sensitive compared with other common sequences. replication slippage may occur for polymerase enzyme during microsatellite amplification and direct sequencing of these pcr products will be challenging for heterozygote samples. so, the aim of this study is to i...

The human fragile-X syndrome, a major cause of inherited mental retardation, is associated with expansion of the trinucleotide repeat GGC:GCC. Repetitive sequences in DNA are subject to slippage during catalysis by DNA polymerases. We characterized the extent of slippage of synthetic GGC:GCC repeats by various DNA polymerases: Taq DNA polymerase, Klenow fragment of DNA polymerase I, DNA Sequenc...

Different amplification protocols were evaluated for use with primer-extension preamplification (PEP). We hypothesized that a protocol known to improve amplification of long DNA fragments would improve efficacy of PEP. Eight DNA samples were preamplified by PCR using different protocols. Treatments consisted of the use of Taq DNA polymerase (T), Taq plus a second polymerase obtained from Pyroco...

To facilitate the investigation of hepatitis B virus (HBV) sequence variation, we recently established a method for functional analysis of PCR-amplified full-length HBV genomes. This study aimed at estimating the number of mutations introduced during amplification of genomes from samples from patients with low levels of viremia and their influence on replication and antigen expression. Wild-typ...

We demonstrate that despite lacking a 3'----5' proofreading exonuclease, the Thermus aquaticus (Taq) DNA polymerase can catalyze highly accurate DNA synthesis in vitro. Under defined reaction conditions, the error rate per nucleotide polymerized at 70 degrees C can be as low as 10(-5) for base substitution errors and 10(-6) for frameshift errors. The frequency of mutations produced during a sin...

A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (> 70 degrees C) were required to significantly relea...

For certain applications of the polymerase chain reaction (PCR), it may be necessary to consider the accuracy of replication. The breakthrough that made PCR user friendly was the commercialization of Thermus aquaticus (Taq) DNA polymerase, an enzyme that would survive the high temperatures needed for DNA denaturation. The development of enzymes with an inherent 3' to 5' exonuclease proofreading...