Efficient DNA sequencing on microtiter plates using dried reagents and Bst DNA polymerase

Area of Science:

• Molecular Biology
• Biochemistry
• Genomic Technologies

Background:

• DNA sequencing is crucial for genetic analysis and research.
• Microtiter plate formats and dried reagents are desirable for automation and storage.
• Enzyme stability during reagent drying is a key challenge for reliable DNA sequencing.

Purpose of the Study:

• To evaluate the suitability of different DNA polymerases for sequencing applications using dried reagents on microtiter plates.
• To optimize drying parameters for enzyme stability and sequencing performance.
• To identify a robust DNA polymerase for automated, high-throughput sequencing workflows.

Main Methods:

• Tested Sequenase, Taq DNA polymerase, and Bst DNA polymerase for DNA sequencing.
• Utilized dried-down reagents on 96-well microtiter plates.
• Investigated parameters to improve the drying process while preserving enzyme activity.

Main Results:

• Sequenase showed poor tolerance to drying, resulting in weak signals and premature termination.
• Taq DNA polymerase yielded good quality sequences but exhibited experimental variability.
• Bst DNA polymerase produced excellent quality sequences and demonstrated stability for over a week at -20°C and overnight at room temperature in a dried state.

Conclusions:

• Bst DNA polymerase is highly stable and effective for DNA sequencing with dried reagents on microtiter plates.
• The developed method using Bst DNA polymerase is well-suited for automated laboratory systems and high-throughput applications.
• This approach enhances the reliability and efficiency of DNA sequencing in robotic workstations.