Abstract
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.
Highlights
Rolling circle amplification (RCA), which is based on the mechanism of the replication of viral genomes, is an isothermal nucleic acid amplification technique that uses two primers, a circularized template and a DNA polymerase with strand displacement activity [1,2,3,4,5]
Our research focused on the acceleration of RCA by microwave heating
We developed a microwave applicator, which avoided the problem of temperature measurement under microwave irradiation and allowed us to implement new reaction conditions that enhanced conventional RCA
Summary
Rolling circle amplification (RCA), which is based on the mechanism of the replication of viral genomes, is an isothermal nucleic acid amplification technique that uses two primers, a circularized template and a DNA polymerase with strand displacement activity [1,2,3,4,5]. RCA efficiently synthesizes many copies of repeated sequences from circular DNA and RNA templates (Fig 1). It detects single nucleotide polymorphisms using a circularized DNA probe called a padlock probe as well as efficiently amplifying bacterial genomes by a hyper-branch method [6,7,8,9,10]. Microwave heating technology is applied to organic and inorganic chemistry using a microwave generator to produce useful effects such as rapid heating, decreased reaction times, and improved product yield and purity[15,16,17,18]. One of the mechanisms of microwave heating in organic chemistry involves heating a low molecular weight compound with a dipole polarization and an ionic conduction
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