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.
Medical Device Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, 701-310 South Korea. jhkim@dgmif.re.kr.
Joong Hyun
JH
Kim
D004252:DNA Mutational Analysis / Q000379:methods*; D006046:Gold; D006801:Humans; D008025:Ligases; D053768:Metal Nanoparticles; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*
C117307:KRAS protein, human; D006046:Gold; D016283:Proto-Oncogene Proteins p21(ras); D008025:Ligases