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 determined directly from total genomic DNA.
Kilger
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Institute of Zoology, University of Munich, PO Box 202126, D-80021 Munich, Germany. kilger@zi.biologie.uni-muenchen.de
Pääbo
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D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase; D003854:Deoxyribonucleotides; D005821:Genetic Techniques; D016678:Genome; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D013698:Templates, Genetic
D003854:Deoxyribonucleotides; D004247:DNA; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase