Dos Protocolos Basados en la RT-PCR para la Identificación de Cadenas con Sentido de Secuencias EST ( Expressed Secuence Tag )

PABLO A. VALDECANTOS; MARTIN E. ARGAÑARAZ; CARLOS M. ABATE; DORA C. MICELI

Tafi del Valle, Tucumán

Jornada; XVIII Jornadas Científicas de la Sociedad de Biología de Tucumán; 2001

Asociación de Biología de Tucumán

In our effort to search for differentially expressed genes in the mammalian oviduct using RAP-PCR, we have identified the Pr14 clone (Accesion #: AF202268), an EST expressed in rat oviducts during the early pregnant stage. Pr14 sequence is similar to several non-related uncharacterized ESTs. In order to know the template strand of Pr14, we designed two different RT-PCR approaches. In the first approach, two cDNA reactions with specific primers were performed; one with primer Pr14A and the other with primer Pr14B. The resulting reverse transcribed reactions were PCR amplified with both specific primers. Only one of the two PCR reactions gave the specific RT-PCR product of the expected size, indicating that the sequence of primer Pr14A is the antisense. In the second ex-perimental approach, a cDNA reaction using oligo dT was per-formed; the reverse transcribed sample was PCR amplified in two reactions using a single specific primer. PCR amplification of the products from linear amplification with both specific primers gave the specific product only in one of the two reactions. The result obtained was according with the first approach. These rapid RT-PCR approaches allowed us the identification of the polarity of the natural RNA of the Pr14 clone, as a first step towards the further characterization of this EST.