PCR free multiple ligase reactions and probe cleavages for the SNP detection of KRAS mutation with attomole sensitivity.

A method to produce multiple ligated primers without PCR for a target DNA containing a single point mutation is presented. A strand displacing hairpin was introduced into the reaction, enabling separation of the ligated primer target DNA duplex without any thermal denaturing process. The ligated product was cycled to allow cleavage of fluorescence labeled substrates for RNase H on gold nanoparticles, leading to target specific fluorescence amplification. As a result, 10 attomoles of target DNA containing a point mutation in the KRAS gene were detected.