Efficient DNA sequencing on microtiter plates using dried reagents and Bst DNA polymerase.
Sequenase, Taq DNA polymerase and Bst DNA polymerase were tested for sequencing of DNA on microtiter plates using dried down reagents. Several parameters were investigated to expedite the drying process while minimizing damage to the enzyme. Sequenase did not tolerate drying very well, and frequently generated sequences with weak signals and many sites of premature termination. With Taq DNA polymerase it was possible to obtain sequences of good quality. However, there was considerable variation of results between experiments and between batches of microtiter plates. Bst DNA polymerase generated sequences of excellent quality. It was stable for more than a week in dried-down state at -20 degrees C and at least overnight at room temperature. The method described here using Bst DNA polymerase is well suited for laboratory robots and workstations that typically employ 96-well microtiter plates.
Earley
J J
JJ
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107.
Kuivaniemi
H
H
Prockop
D J
DJ
Tromp
G
G
D001331:Automation; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase; D005821:Genetic Techniques; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007202:Indicators and Reagents; D008832:Microchemistry; D017422:Sequence Analysis, DNA / Q000379:methods*; D019914:Taq Polymerase
D007202:Indicators and Reagents; D004247:DNA; D019914:Taq Polymerase; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase