Inosine 5'-triphosphate can dramatically increase the yield of NASBA products targeting GC-rich and intramolecular base-paired viroid RNA.
Nucleic acid sequence-based amplification (NASBA) according to the standard protocol failed to amplify cRNA of viroids, probably because of their GC-rich and intramolecular base-paired structure. However, NASBA in the presence of inosine 5'-triphosphate successfully amplified the cRNAs to viroids in total nucleic acid extracts from citrus plants. As sequence specificity of the cRNA to viroids was confirmed by northern analysis, the amplification and fidelity of cRNAs are sufficient for the sensitive and specific detection of viroids.
Nakahara
K
K
Department of Agrobiology and Bioresources, Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Hataya
T
T
Uyeda
I
I
D001482:Base Composition; D001483:Base Sequence; D003596:Cytosine; D017931:DNA Primers; D006147:Guanine; D007293:Inosine Triphosphate / Q000032:analysis* / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D010942:Plant Viruses / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D014772:Viroids / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D012367:RNA, Viral; D007293:Inosine Triphosphate; D006147:Guanine; D003596:Cytosine