RESUMEN
The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This promoter sequence is made double-stranded (hence functional) by DNA polymerase, allowing T7 RNA polymerase to generate a target-dependent RNA signal which is measured by an enzyme-linked oligosorbent assay (ELOSA). The sequence of the RNA signal is always the same, regardless of the original target sequence. The SMART assay was successfully tested in model systems with several single-stranded synthetic targets, both DNA and RNA. The assay could also detect specific target sequences in both genomic DNA and total RNA from Escherichia coli. It was also possible to generate signal from E.coli samples without prior extraction of nucleic acid, showing that for some targets, sample purification may not be required. The assay is simple to perform and easily adaptable to different targets.
Asunto(s)
ADN/genética , Reacción en Cadena de la Polimerasa/métodos , ARN/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , ADN/química , Sondas de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli/genética , Humanos , Conformación de Ácido Nucleico , Oligonucleótidos/genética , ARN/química , ARN Bacteriano/química , ARN Bacteriano/genéticaRESUMEN
The ability of a blocked or overshadowed conditioned stimulus (CS) to serve as (a) blocker or (b) a 2nd-order reinforcer in Pavlovian fear conditioning was tested in 152 albino rats. CS-evoked suppression of barpressing for food was the index of conditioned fear. Experiments 1 and 2 showed that an overshadowed CS was weakened in its ability to serve as a blocker. In Experiment 2, a blocked CS was similarly weakened. Experiment 3 showed that an overshadowed and blocked CS was weakened in its ability to serve as a 2nd-order reinforcer. Experiments 4 and 5 failed to restore the blocking ability of blocked (Experiment 4) or overshadowed (Experiment 5) CSs by extinguishing the CSs that had blocked or overshadowed them. Results favor a learning-deficit view of blocking and overshadowing.
Asunto(s)
Atención , Condicionamiento Clásico , Miedo , Refuerzo en Psicología , Animales , Conducta Apetitiva , Aprendizaje por Asociación , Extinción Psicológica , Femenino , Masculino , RatasRESUMEN
In a sample of 208 Holtzman-descended albino rats, we found evidence with 4 measures of conditioning (freezing, defecation, side crossing, and nose crossing) that a single 2-s, 1.0-mA immediate shock could condition fear to a context (Experiments 1, 2, and 4). When we reduced the shock intensity to 0.5 mA, we obtained a complete immediate-shock conditioning deficit according to all measures in Experiment 3 and to all but the defecation measure in Experiment 4. Results suggest two conclusions: (a) Differences in shock potency between laboratories may help explain discrepant findings about whether immediate shock supports contextual conditioning; (b) theories of contextual conditioning need a mechanism that permits that conditioning to result from immediate shock.