Sensitive and specific detection of carcinoembryonic antigen cDNA using the hot start polymerase chain reaction...

This study evaluated the specificity and sensitivity of hot start polymerase chain reaction (RT-PCR) for identification of carcinoembryonic antigen (CEA) cDNA. Hot Start Storage and Reaction Tubes provide a simple mechanism for synchronizing hot start amplifications without the need for manual intervention. A wax barrier uses a layer of solid wax to separate the retained reagents and the test sample from the bulk of the reagents until the first heating step of automated thermal cycling melts the wax and convectively mixes the two aqueous layers. Wax-mediated hot start PCR greatly increases the specificity and sensitivity of amplifying CEA cDNA. 12 out of 12 samples were positive for CEA cDNA from 10 CEA mRNA positive tumor cells in 10(7) normal cells by hot start CEA-PCR, and 8 out of these samples were positive for CEA cDNA by CEA-PCR without hot start. The hot start PCR showed the single specific product band, but PCR without hot start showed the specific product band and the other non-specific product band. We conclude that the hot start PCR technique could sensitively and specifically detect CEA cDNA after reverse transcription of CEA mRNA and minimize primer dimers by amplification.