Controlled Microwave Heating Accelerates Rolling Circle Amplification.

Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same.

Estimation of DNA polymerase for improvement of rolling circle amplification.

Rolling Circle Amplification (RCA) is a technique to amplify the oligoncleotides containing tandemly repetitive sequence motif. Various repetitive sequences of which the motif unit is the circular template can be synthesized by extending the primer. Additionally, RCA need not change the temperature regulatory processes like PCR, but RCA has the gene amplification efficiency similar to PCR. Although, it contains the problem of the sequence confirmation and the activity of polymerase as the DNA polymerase I. We comparatively estimated of the various polymerases for the improvement of the RCA. Five DNA polymerases were estimated for RCA.

Microwave assisted rolling circle amplification.

The Rolling Circle Amplification is an effective method of DNA amplification having tandem repeated sequences. Firstly, the template sequence is designed to form a circular structure by the annealing with the primer. The thermostable polymerase is catalyzed to primer and circular template DNA. As a result, tandem repetitive olignucleotide of circular template sequence can be obtained. In our recent study, Bst DNA polymerase catalyzed RCA reaction had been elucidated that the oligonucleotides with tandem repeated sequence were obtained as the ladder band and amplified the enough amounts of DNA within short time. On the other hand, we also focused on the microwave technology for advance to the various chemical and biological reactions. In this study, the microwave was irradiated to RCA reaction on controlling the temperature.