Ⅲ.Chemistry

東京大学Editor : Tazaki, Kazue |田崎, 和

Controlled Microwave Heating Accelerates Rolling Circle Amplification

<div><p>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.</p></div

Electrophoresis and fluorescence intensity of DNA synthesized by four DNA polymerases under conventional and microwave heating.

<p>MW = microwave. (a) <i>Bst</i> DNA polymerase-LF, 10–60-min rolling circle amplification (RCA) and10–30-min microwave-assisted (MW)-RCA. (b) <i>Bst</i> DNA polymerase, 10–60-min RCA and 10–30-min MW-RCA (c) <i>Csa</i> DNA polymerase, 10–90-min RCA and 10–30-min MW-RCA. (d) 96–7 DNA polymerase, 10–120-min RCA and 10–30 min MW-RCA.</p

The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

<p>The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.</p

Comparison of temperatures of rolling circle amplification components from 13°C to 60°C using conventional (a) and microwave heating (b).

<p>Conventional heating was conducted in a 60°C heating block.</p

Temperature profile (a), electric power profile (b), and frequency profile (c) of MW-RCA using <i>Bst</i> DNA polymerase-LF at 60°C.

<p>Temperature profile (a), electric power profile (b), and frequency profile (c) of MW-RCA using <i>Bst</i> DNA polymerase-LF at 60°C.</p

Microwave-assisted rolling circle amplification reaction mixtures containing 4-fold increased concentrations of one component.

<p>(a) Agarose gel electrophoresis results. (b) Fluorescence intensity of SYBR Green I. C = conventional, MW = microwave.</p