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Nucleic Acids Research, 1988, Vol. 16, No. 8 3487-3496
© 1988

Articles

T7 DNA polymerase in automated dideoxy sequencing

Tom Kristensen1, Hartmut Voss, Christian Schwager, Josef Stegemann, Brian Sproat and Wilhelm Ansorge*

European Molecular Biology Laboratory Heidelberg, FRG 1Research Institute for Internal Medicine, University of Oslo Norway

*To whom correspondence should be addressed

Received March 3, 1988. Accepted March 17, 1988.

T7 DNA polymerase with chemically inactivated 3'-5' exonuclease activity, as well as unmodified T7 DNA polymerase, were used for sequencing by the dideoxy method in an automated system with fluorescence labelled primer and on-line detection of laserexcited reaction products. An analysis of signal intensity variations in the C track revealed that low C signals were usually preceded by a T in the sequence. This effect was modified by surrounding nucleotides. Signal intensities were more uniform with T7 polymerase than with the Klenow fragment of DNA polymerase I. Some sequences ambiguous with the Klenow enzyme could easily be evaluated with the T7 enzyme. One sequence could only be read by the unmodified T7 polymerase, while both the Klenow fragment and the chemically modified T7 enzyme gave uninterpretable data.


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