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Journal of Biochemistry Advance Access originally published online on August 24, 2007
Journal of Biochemistry 2007 142(2):273-281; doi:10.1093/jb/mvm138
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© 2007 The Japanese Biochemical Society.

Highly Efficient Isothermal DNA Amplification System Using Three Elements of 5'-DNA-RNA-3' Chimeric Primers, RNaseH and Strand-displacing DNA Polymerase

Hiroyuki Mukai1, Takashi Uemori2, Osamu Takeda1, Eiji Kobayashi2, Junko Yamamoto1, Kazue Nishiwaki1, Tatsuji Enoki2, Hiroaki Sagawa2, Kiyozo Asada2,* and Ikunoshin Kato2

1Products Development Center, Takara Bio Inc., 2257, Noji, Kusatsu, Shiga 525-0055; and 2Biotechnology Research Laboratories, Takara Bio Inc., Seta 3-4-1 Otsu, Shiga 520-2193, Japan

*To whom correspondence should be addressed. Tel: +81 77-543-7234, Fax: +81 77-543-7295, E-mail: asadak{at}takara-bio.co.jp

Received January 10, 2007; Accepted June 12, 2007


  Abstract

We developed an efficient method of isothermally amplifying DNA termed ICAN, Isothermal and Chimeric primer-initiated Amplification of Nucleic acids. This method allows the amplification of target DNA under isothermal conditions at around 55°C using only a pair of 5'-DNA-RNA-3' chimeric primers, a thermostable RNaseH and a DNA polymerase with strong strand-displacing activity. ICAN is capable of amplifying DNA at least several times greater than the amount produced with PCR by increasing primer concentration. This method would be applicable for on-site DNA detection including gene diagnosis, and would also be suitable for ‘real time’ detection when combined with a cycling probe.

Key Words: chimeric primer, detection, DNA polymerase, isothermal DNA amplification, RNaseH

Abbreviations: CP4 EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium sp. CP4; CSVd, chrysanthemum stunt viroid; HDA, helicase-dependent amplification; ICAN, isothermal and chimeric primer-initiated amplification of nucleic acids; LAMP, loop-mediated isothermal amplification of DNA; RCA, rolling-circle amplification; SDA, strand displacement amplification; Tli RNaseH, Thermococcus litoralis RNaseH; TMA, transcription-mediated amplification


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