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

摘要

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.