Katsunori Ikeda

Development of a novel method for operating magnetic particles, Magtration Technology, and its use for automating nucleic acid purification

Magnetic particles are useful for simple and efficient nucleic acid extraction. To achieve fully automated nucleic acid extraction and purification using magnetic particles, a new method for operating magnetic particles, Magtration Technology, was developed. In this method, magnetic separation is performed in a specially designed disposable tip. This enables high recovery of magnetic particles with high reproducibility. The features of this technology are (i) a simple mechanism for process control and (ii) flexible software to enable adaptation to commercially available reagents. Automated instruments based on Magtration Technology were developed and used for nucleic acid extraction. Total DNA, total RNA and plasmids were purified by Magtration Technology at an efficiency comparable to that of manual methods.

Construction and characterization of N-terminally truncated DNA polymerase from Thermus thermophilus

Abstract Various plasmids harboring the truncated DNA polymerase gene ( polA ) from Thermus thermophilus HB8 ( Tth polymerase) were constructed. The most thermostable Tth Δ NF 2 polymerase [the gene product of polA Δ NF 2, which lacked a 751-bp region (region flanked by initiation codon and Fsp I site in the polA gene)] was selected, and purified from the recombinant Escherichia coli . SDS polyacrylamide gel electrophoresis revealed that the molecular weight of the Tth Δ NF 2 polymerase is 58–61 kDa, which is approximately 30 kDa smaller than that of the wild-type enzyme. The specific activity of the 5′-to-3′ polymerization of the Tth Δ NF 2 polymerase was 63% of that of the Tth polymerase. However, no 5′-to-3′ exonuclease activity was detected in this mutant enzyme (less than 1% of the specific activity of wild-type enzyme). The activities of the wild-type and mutant enzymes were maximal at 75°C. Approximately 50% of the enzyme activity was retained even after heat treatment of the Tth Δ NF 2 polymerase at 70°C for 2 h, but the thermostability of the mutant enzyme was slightly lower than that of the wild-type enzyme. Both the Tth Δ NF 2 and Tth polymerases were capable of non-templated addition of deoxyribonucleotide to a 3′-hydroxyl group of blunt-ended DNA.