Investigation of the molecular mechanism of ICAN, a novel gene amplification method.
Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (ICAN) allows the amplification of target DNA under isothermal conditions at around 55 degrees 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 analysing the nicking site of RNaseH, behaviour 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.
Uemori
Takashi
T
Biotechnology Research Laboratories, Takara Bio Inc., Seta 3-4-1 Otsu, Shiga 520-2193, Japan.
Mukai
Hiroyuki
H
Takeda
Osamu
O
Moriyama
Mariko
M
Sato
Yoshimi
Y
Hokazono
Shigekazu
S
Takatsu
Nariaki
N
Asada
Kiyozo
K
Kato
Ikunoshin
I
D053904:DNA Breaks, Single-Stranded; D017931:DNA Primers / Q000737:chemistry; D008954:Models, Biological; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016914:Ribonuclease H / Q000378:metabolism
D017931:DNA Primers; D016914:Ribonuclease H