Direct PCR Sequencing with Denaturants (Formamide)

The advance of Taq -based polymerase chain reaction (PCR) technology (1 –3 ) has had a tremendously positive impact on biomedical research. The combination of PCR and sequencing further revolutionized biological research (3 , 4 ). Sequence amplification technology has provided a speedy and effective alternative to work-intensive gene cloning strategies. In addition, small quantities of precious clinical samples that were clearly insufficient for definitive structural analysis by conventional cloning techniques are now sufficient for such analysis using PCR cloning and sequencing methods (5 ,6 ). Using these techniques, the genetic changes that exert a dominant effect on the phenotype of neoplastic cells have been brought to light during the past five years (7 –9 ). In addition, many novel genes that govern the proliferation and differentiation of cells have been identified and cloned using redundant primers (10 , 11 ). These achievements have greatly advanced our understanding of the regulatory machinery that governs cell growth. The understanding of how these changes in cellular regulation contribute to tumorigenesis has opened the doors to the development of genetic therapy for human diseases.