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Nucleic Acids Research, 1989, Vol. 17, No. 23 9613-9620
© 1989

MOLECULAR BIOLOGY

Incomplete primer extension during in vitro DNA amplification catalyzed by Taq polymerase; exploitation for DNA sequencing

David B. Olsen and Fritz Eckstein

Max-Planck Institut für Experimentelle Medizin, Abteilung Chemie Hermann-Rein Strasse 3, D-3400 Göttingen, FRG

Received September 13, 1989. Revised November 3, 1989. Accepted November 3, 1989.

Polyacrylamide gel electrophoresis of DNA fragments obtained by the polymerase chain reaction using Taq polymerase revealed the presence of multiple fragments shorter than the expected product. These abortive extension products were observed even when analysis by agarose gel electrophoresis showed only a single band. The production of prematurely terminated fragments can be exploited for the sequencing of PCR products if phosphorothioate groups are incorporated base specifically during the reaction in the presence of two oligonucleotide primers, one of which is 5'-32P-labeled The addition of snake venom phosphodiesterase to the reaction mixture after completion of the amplification cycles digests each fragment from the 3'-end to a phosphorothioate group so that the sequence can be read by polyacrylamide gel electrophoresis.


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