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Automated cycle sequencing with Taquenase: protocols for internal labeling, dye primer and "doublex" simultaneous
1Pharmacia Biotech, Milwaukee, WI, USA.
Biotechniques
|August 1, 1997
Summary
This study introduces Taquenase, a modified DNA polymerase, for enhanced automated DNA sequencing. It enables accurate sequencing of up to 2000 bases with high efficiency using internal labeling and primer walking methods.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Automated cycle sequencing is crucial for DNA analysis.
- Existing thermostable DNA polymerases have limitations in incorporating labeled nucleotides.
- Efficient sequencing of large DNA constructs like PACs requires improved enzyme performance.
Purpose of the Study:
- To describe automated cycle sequencing protocols using Taquenase.
- To evaluate the performance of Taquenase in DNA sequencing applications.
- To demonstrate the feasibility of high-accuracy, long-read sequencing with Taquenase.
Main Methods:
- Development of automated cycle sequencing protocols for internal labeling, dye primer, and "doublex" sequencing.
- Utilizing Taquenase, a genetically modified thermostable DNA polymerase.
- Performing primer walking on Pl-derived artificial chromosome (PAC) constructs.
- Comparing Taquenase with other modified Taq DNA polymerases.
Main Results:
- Taquenase efficiently incorporates fluorescently labeled dNTPs (fluorescein isothiocyanate, TexasRed, Cy5).
- Uniform and unambiguous signals were obtained up to the resolution limit of sequencing gels.
- Successful primer walking on 130-kb PAC inserts.
- Achieved sequencing of up to 2000 bases with >99% accuracy in a single reaction.
Conclusions:
- Taquenase offers superior performance in cycle sequencing compared to other thermostable polymerases.
- The enzyme's N-terminal deletion is key to its efficient incorporation of labeled dNTPs.
- Taquenase enables accurate simultaneous sequencing of both DNA strands, facilitating large-scale genomic analysis.