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Direct cycle sequencing with delta Taq DNA polymerase

J Fan¹, R S Ranu

¹Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins 80523, USA.

DNA Sequence : the Journal of DNA Sequencing and Mapping

|January 1, 1997

Summary

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Delta Taq DNA polymerase, a modified enzyme lacking 5'-->3'-exonuclease activity, is effective for direct cycle sequencing. This versatile enzyme enables DNA sequencing from various PCR-amplified and single-stranded templates.

Area of Science:

Molecular Biology
Biochemistry
Genetics

Background:

Standard Taq DNA polymerase is widely used in molecular biology.
The 5'-->3'-exonuclease activity of standard Taq polymerase can interfere with certain applications.
Delta Taq DNA polymerase is a genetically modified variant lacking this exonuclease activity.

Purpose of the Study:

To evaluate the utility of delta Taq DNA polymerase for direct cycle sequencing.
To determine optimal conditions for sequencing using delta Taq DNA polymerase.

Main Methods:

Direct cycle sequencing using delta Taq DNA polymerase.
Utilizing various DNA templates including PCR-amplified DNA, plasmid DNA, and single-stranded DNA.
Optimization of primer-to-template ratios and cycle numbers.

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Main Results:

Delta Taq DNA polymerase successfully performed direct cycle sequencing across diverse template types.
Optimal primer-to-template ratios and cycle numbers were established for reliable sequence data.
The enzyme demonstrated efficacy with asymmetrically amplified, double-stranded, cloned, and single-stranded DNA templates.

Conclusions:

Delta Taq DNA polymerase is a versatile and effective tool for direct cycle DNA sequencing.
The enzyme's lack of 5'-->3'-exonuclease activity facilitates direct sequencing applications.
This polymerase offers a robust alternative for DNA sequence determination.