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Direct cycle sequencing with delta Taq DNA polymerase
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
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.
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.