Rapid DNA chemical ligation for amplification of RNA and DNA signal.
Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate--iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.
Abe
Hiroshi
H
Nano Medical Engineering Laboratory, Discovery Research Institute, RIKEN, 2-1, Hirosawa, Wako-Shi, Saitama, 351-0198 Japan. h-abe@riken.jp
Kondo
Yuko
Y
Jinmei
Hiroshi
H
Abe
Naoko
N
Furukawa
Kazuhiro
K
Uchiyama
Atsushi
A
Tsuneda
Satoshi
S
Aikawa
Kyoko
K
Matsumoto
Isamu
I
Ito
Yoshihiro
Y
D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D007461:Iodoacetates / Q000138:chemical synthesis / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009691:Nucleic Acid Denaturation; D009946:Organothiophosphorus Compounds / Q000138:chemical synthesis / Q000737:chemistry; D012313:RNA / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics
D007461:Iodoacetates; D009946:Organothiophosphorus Compounds; D012313:RNA; D004247:DNA