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Journal of Biochemistry Advance Access published online on August 24, 2007
Journal of Biochemistry, 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

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

#To whom correspondence should be addressed: Dr. Kiyozo Asada, (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


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