Multiple chemical ligation under thermal cycle.
Enzymatic ligation methods are useful in diagnostic detection of DNA sequence. Here we describe the investigation of nonezymatic phosphorothioate-iodoacetyl DNA chemical ligation as a method for detection and identification of RNA and DNA. Specificity of ligation on DNA target is shown to yield discrimination of single point mutation as a drop in two magnitude. Although enzymatic ligation shows very low activity for RNA target, this reaction is found to be very efficient on RNA target. This chemical ligation with RNA target completes 70% within a few seconds, which equal or overcome ligase enzyme-mediated ligation with DNA target. The reaction is also shown to exhibit a significant level of signal amplification under thermal cycle for short time. Further, we found recently that ligation fidelity changed in function of chemical reactivity of probe. This trend was systematically investigated.
Kondo
Yuko
Y
Nanomedical Engineering Laboratory, Discovery Research Institure, Riken, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. yukok@riken.jp
Abe
Hiroshi
H
Jinmei
Hiroshi
H
Abe
Naoko
N
Aikawa
Kyoko
K
Matsumoto
Isamu
I
Ito
Yoshihiro
Y
D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes / Q000737:chemistry*; D007461:Iodoacetates / Q000737:chemistry; D007700:Kinetics; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000032:analysis*; D013696:Temperature; D013698:Templates, Genetic
D015342:DNA Probes; D007461:Iodoacetates; D054735:Phosphorothioate Oligonucleotides; D012313:RNA