Manual and Automated Direct Sequencing of Product Generated by the Polymerase Chain Reaction

Identification of point mutations has been facilitated by a number of techniques, including transfection assays, oligonucleotide hybridization, electrophoretic migration of heteroduplexes, RNase mismatch analysis, direct sequencing, and DNA-polymerase catalyzed amplification. The large number of available techniques emphasizes the importance of developing rapid and reliable methods to identify molecular changes in genes. To date, we have concentrated on exploiting DNA-polymerase catalyzed amplification methods (1 ,2 ) in conjunction with direct manual and automated DNA sequencing to detect point mutations in the dihydrofolate reductase (DHFR) gene of methotrexate-resistant cells.