DNA Structure Dependence of DNA Binding by Escherichia coli and Thermus aquaticus DNA Polymerase I

production of thermus aquaticus dna polymerase in escherichia coli

Salt dependence of DNA binding by Thermus aquaticus and Escherichia coli DNA polymerases.

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...

Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli.

DNA polymerase from Thermus aquaticus has become a common reagent in molecular biology because of its utility in DNA amplification and DNA sequencing protocols. A simplified method is described here for isolating the recombinant Taq enzyme after overproduction in Escherichia coli. Purification requires 8 to 10 h and entails heat treating and clearing the E. coli lysate, followed by precipitatio...

High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase

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...

Cloning and Expression of Thermus Aquaticus DNA Polymerase Gene, Using a Thermo-Inducible Expression Vector

DNA polymerase gene from Thermus aquaticus strain YT1 was amplified using VENTTM DNA po-lymerase and cloned under the control of X.PR promoter and expression was induced by a shift in tern perature. The culture was then sonicated, and after centrifugation the lysate was treated with poly‌ethyleneimine followed by a salting-out step. Finally the protein was precipitated with ammonium sulfate and...