Tailing cDNAs with terminal deoxynucleotidyl transferase in RT-PCR assays to identify ribozyme cleavage products.
Polytailing a cDNA with terminal deoxynucleotidyltransferase (TdT) results in the addition of a homopolymeric sequence at its 3'-end. Here we describe the use of tailing in competitive RT-PCR assays to evaluate cleavage efficiency of ribozymes. Using a system that perfectly mimics intracellular cleavage, we were able to detect as few as 1% of cleaved moieties. Furthermore, employing primers overlapping the junction between tails and the cleaved RNA moiety in non-competitive assays, the sensitivity of the method could be improved to <10 fg. Using the latter protocol and reactions employing a trans -acting hairpin ribozyme targeting the nucleocapsid mRNA of the mumps virus, we were able to demonstrate ribozyme-induced cleavage.
Albuquerque-Silva
J
J
Applied Genetics, University of Brussels, Brussels, Belgium.
Houard
S
S
Bollen
A
A
D001483:Base Sequence; D004253:DNA Nucleotidylexotransferase / Q000737:chemistry*; D017931:DNA Primers; D018076:DNA, Complementary; D006868:Hydrolysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D016337:RNA, Catalytic / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D016337:RNA, Catalytic; D004253:DNA Nucleotidylexotransferase