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Nucleic Acids Research, 1988, Vol. 16, No. 21 9947-9959
© 1988

CHEMISTRY

Direct sequencing of polymerase chain reaction amplified DNA fragments through the incorporation of deoxynucleoside {alpha}-thiotriphosphates

Kay L. Nakamaye+, Gerald Gish, Fritz Eckstein* and Hans-Peter Vosberg1

Max-Planck-Institut für Experimentelle Medizin Abteilung Chemie, Hermann-Rein-Strasse 3, D-3400 Göttingen 1Max-Planck-Institut für Medizinische Forschung Jahnstrasse 29, D-6900 Heidelberg, FRG

*To whom requests for reprints should be sent

Received September 5, 1988. Accepted October 7, 1988.

The direct sequencing of DNA generated by the polynucleotide chain reaction, via the incorporation of phosphorothioate nucleotides and followed by treatment with an alkylating reagent that cleaves specifically at the phosphorothioate positions, is described. The Taq polymerase used in the amplification reaction incorporates the Sp-diastereomer of the deoxynucleoside 5'-0-(1-thiotriphosphates) as efficiently as the natural nucleotides. Chemical degradation of the phosphorothioate-containing DNA fragment can be performed with either 2-iodoethanol or 2,3-epoxy-1-propanol. The higher reactivity of 2,3-epoxy-1-propanol allows less reagent to be used to obtain the same amount of degradation as with 2-iodoethanol.

+ Present address: Department of chemistry, Gonzaga University, Spokane, WA 99203, USA


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