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Journal of Biochemistry Advance Access published online on August 24, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm137
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© 2007 The Japanese Biochemical Society

Investigation of the molecular mechanism of ICAN, a novel gene amplification method

Takashi Uemori1, Hiroyuki Mukai2, Osamu Takeda2, Mariko Moriyama1,*, Yoshimi Sato1, Shigekazu Hokazono1, Nariaki Takatsu2, Kiyozo Asada1,# and Ikunoshin Kato1

Takara Bio Inc. 1Biotechnology Research Laboratories, Seta 3-4-1 Otsu, Shiga, 520-2193, Japan, 2Products Development Center, 2257, Noji, Kusatsu, Shiga, 525-0055, 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 April 3, 2007; Accepted June 12, 2007


  Abstract

Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (ICAN) allows the amplification of target DNA under isothermal conditions at around 55°C using only a pair of 5’-DNA-RNA-3’ chimeric primers, thermostable RNaseH, and a DNA polymerase with strand- displacing activity [H. Mukai et al. J. Biochemistry, in the preceding paper in this issue]. Here we elucidated the mechanism of ICAN by analyzing the nicking site of RNaseH, behavior of chimeric primers, and extension products. We found that the ICAN reaction was composed of two unique mechanisms, multi-priming and template-switching, that were responsible for the highly efficient amplifying capability of ICAN. The simultaneous occurrence of two types of reactions, one based on multi-priming and the other based on template-switching, is likely to drive the DNA amplification in ICAN.

Key Words: ICAN, multi-priming, RNaseH, strand displacement, template-switching

*Present address: Cutaneous Biology Research Center, Massachusetts General Hospital (Building 149), 13th Street, Charlestown, MA 02129, USA


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