Direct DNA sequence determination from total genomic DNA

It is possible to perform a combined amplification and sequencing reaction (‘DEXAS’) directly from complex DNA mixtures by using two thermostable DNA polymerases, one that favours the incorporation of deoxynucleotides over dideoxynucleotides, and one which has a decreased ability to discriminate between these two nucleotide forms. During cycles of thermal denaturation, annealing and extension, the former enzyme primarily amplifies the target sequence whereas the latter enzyme primarily performs a sequencing reaction. This method allows the determination of single-copy nuclear DNA sequences from amounts of human genomic DNA comparable to those used to amplify nucleotide sequences by the polymerase chain reaction. Thus, DNA sequences can be easily Fig. 1 . Schematic illustration of DEXAS and the ‘two polymerase’ concept. determined directly from total genomic DNA. Each reaction tube contains (among other components) template DNA, one labeled and one unlabeled primer, dNTPs, one of the four ddNTPs, one DNA.