Highly efficient isothermal DNA amplification system using three elements of 5'-DNA-RNA-3' chimeric primers, RNaseH and strand-displacing DNA polymerase.
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 degrees 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.
Mukai
Hiroyuki
H
Products Development Center, Takara Bio Inc., 2257, Noji, Kusatsu, Shiga 525-0055, Japan.
Uemori
Takashi
T
Takeda
Osamu
O
Kobayashi
Eiji
E
Yamamoto
Junko
J
Nishiwaki
Kazue
K
Enoki
Tatsuji
T
Sagawa
Hiroaki
H
Asada
Kiyozo
K
Kato
Ikunoshin
I
D004247:DNA / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012313:RNA / Q000737:chemistry*; D016914:Ribonuclease H / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
D017931:DNA Primers; C034477:RNA primers; D012313:RNA; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H