RESUMO
Amplification of specific gene target sequences has become a routine molecular procedure in a variety of laboratories. When coupled with either a direct or indirect method of detecting amplified product, in situ amplification offers an extremely powerful investigative tool. We describe a protocol for a localized in situ amplification (LISA) reaction that includes tissue-culture cloning rings and allows for the amplification of gene target sequences in specific regions of paraffin-embedded tissue sections. Digoxigenin-11-dUTP was added to the amplification reaction and thus incorporated into the amplified products, providing a mechanism by which direct nonisotopic detection could be performed. To demonstrate the approach, LISA was performed on known positive Pneumocystis carinii rat lung tissues, with primers specific for the P. carinii rRNA gene sequence.
Assuntos
Hibridização In Situ/métodos , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Eletroforese em Gel de Ágar , Pulmão/microbiologia , Dados de Sequência Molecular , Pneumocystis/genética , Infecções por Pneumocystis/microbiologia , RNA Fúngico/análise , RNA Ribossômico/análise , RatosRESUMO
A retrospective analysis of 47 cases of clinical stage IB cervical carcinoma radiologically staged with computed tomography (CT) was undertaken. There were no cases where CT provided information which altered staging. In 5 cases, CT suggested extension of disease beyond the cervix which was not confirmed surgically or with other staging procedures. In 3 cases, one or more normal-sized obturator or internal iliac nodes with metastatic disease were discovered at surgery, but the high common iliac and periaortic nodes were disease free. We conclude that routine use of CT in patients with clinical stage IB cervical carcinoma is not warranted unless the patient's body habitus precludes accurate physical examination.
