Tailing cDNAs with terminal deoxynucleotidyl transferase in RT-PCR assays to identify ribozyme cleavage products.
Nucleic Acids Res
; 26(13): 3314-6, 1998 Jul 01.
Article
en En
| MEDLINE
| ID: mdl-9628937
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Reacción en Cadena de la Polimerasa
/
ARN Catalítico
/
ADN Nucleotidilexotransferasa
Idioma:
En
Revista:
Nucleic Acids Res
Año:
1998
Tipo del documento:
Article
País de afiliación:
Bélgica
Pais de publicación:
Reino Unido