Isothermal DNA Amplification by a Novel and Non-Ubiquitous Thermus Polymerase A
Sigridur Hjorleifsdottir, Thorarinn Blöndal, Arnthor Evarsson, Ólafur H. Fridjónsson, Sveinn Ernstsson, Gudmundur O. Hreggvidsson, Einar Mäntylä, Paul J. Rothwell and Jakob K. Kristjansson
Affiliation: Prokazyme Ltd., Vínlandsleid 14, P.O. Box 10165, IS-130 Reykjavik, Iceland.
Keywords: Isothermal DNA amplification, strand displacement, thermophilic DNA polymerase, Thermus.
A novel and non-ubiquitous thermostable DNA polymerase in Thermus antranikianii was expressed in E. coli,
isolated and biochemically characterized. The enzyme here referred to as Thermophi, has a C-terminal polymerase domain
and a proofreading 3′→5′ exonuclease domain, but lack the 5′→3′ exonuclease domain. The corresponding gene is
apparently only found in some but not all Thermus strains. The initial rate of specific activity of this polymerase on nicked
DNA was about 360,000 U/mg protein. The optimum activity was found at 55 °C, pH 8.5 and 1.5 mM Mg+2. The
polymerase was stable at 70 °C and lost 50% of its activity after 5 min at 85 °C, but could be stabilized above 80 °C by
addition of 0.5 M L-proline. A pronounced strand-displacement activity was indicated by the large amount of DNA
produced by the enzyme after an overnight, isothermal incubation in presence of hexamer primers. Both single and double
stranded DNA was isothermally amplified by the enzyme. The amplified DNA was large and apparently highly branched
material and composed of both single and double stranded DNA. The produced material could be partly digested by T7
enonuclease I but it was difficult to cut with common restriction enzymes. Amplification of selected genes from dilute
samples was successfully demonstrated with the human β-actin gene. Good amplification was also found with 5
microsatellite markers from salmon DNA. Thermophi amplifies DNA by orders of magnitude but upon extended reaction
time the DNA becomes very large and highly branched. It is composed of both single and double strands and then
correctly amplified sequences only represent about 10-20% of the total DNA, and long stretches of TATATA repeats
frequently occur in the amplified DNA.
Rights & PermissionsPrintExport