DNA binding of the Type 1 DNA polymerase from Thermus aquaticus (Taq polymerase) and its Klentaq large fragment domain have been studied as a function of temperature. Equilibrium binding assays were performed from 5 to 70 degrees C using a fluorescence anisotropy assay and from 10 to 60 degrees C using isothermal titration calorimetry. In contrast to the usual behavior of thermophilic proteins ...

Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clinical and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymerase, since mutational alteration of the polymerase can overcome the inhibition to the extent that no DNA purification is now required. An N-terminal deletion (Klentaq1) is some 10-100-fold inhibi...

PCR co-amplification of two distinct HIV1 tat gene sequences lead to the formation of recombinant DNA molecules. The frequency of such recombinants, up to 5.4% of all amplified molecules, could be decreased 2.7 fold by a 6 fold increase in Taq DNA polymerase elongation time. Crossover sites mapped essentially to three discrete regions suggesting specific Taq DNA polymerase pause or termination ...

UNLABELLED PREMISE OF THE STUDY Secondary metabolites often inhibit PCR and sequencing reactions in extractions from plant material, especially from silica-dried and herbarium material. A DNA polymerase that is tolerant to inhibitors improves PCR results. • METHODS AND RESULTS A novel DNA amplification system, including a DNA polymerase engineered via directed evolution for improved tolera...

As the sole renal Na,K-ATPase isozyme, the a, Na,K-ATPase accounts for all active transport of Na throughout the nephron. This role in renal Na reabsorption and the primacy of the kidney in hypertension pathogenesis make it a logical candidate gene for salt-sensitive genetic hypertension. An adenine (A)—>thymine (T) transversion, resulting in the substitution of glutamine with leucine and assoc...

We investigated three probe design strategies used in quantitative polymerase chain reaction (PCR) for sensitivity in detection of the PCR amplicon. A plasmid with a 120-bp insert served as the DNA template. The probes were TaqMan, conventional molecular beacon (MB), and shared-stem molecular beacon (ATssMB and GCssMB). A shared-stem beacon probe combines the properties of a TaqMan probe and a ...

The RFLP/PCR approach (restriction fragment length polymorphism/polymerase chain reaction) to genotypic mutation analysis described here measures mutations in restriction recognition sequences. Wild-type DNA is restricted before the resistant, mutated sequences are amplified by PCR and cloned. We tested the capacity of this experimental design to isolate a few copies of a mutated sequence of th...

DNA binding properties of the Type 1 DNA polymerases from Thermus aquaticus (Taq, Klentaq) and Escherichia coli (Klenow) have been examined as a function of [KCl] and [MgCl(2)]. Full-length Taq and its Klentaq "large fragment" behave similarly in all assays. The two different species of polymerases bind DNA with sub-micromolar affinities in very different salt concentration ranges. Consequently...