DIRECT DNA-SEQUENCE DETERMINATION FROM TOTAL GENOMIC DNA

It is possible to perform a combined amplification and sequencing reac tion ('DEXAS') directly from complex DNA mixtures by using two thermos table DNA polymerases, one that favours the incorporation of deoxynucl eotides 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 prima rily 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 polym erase chain reaction. Thus, DNA sequences can be easily determined dir ectly from total genomic DNA.