29] Modified T7 DNA polymerase for DNA sequencing

Abstract

Publisher Summary This chapter discusses various modified T7 DNA polymerase for DNA sequencing. When bacteriophage T7 infects its Escherichia coli host, it directs the production of a phage-specific DNA polymerase. This enzyme is a two subunit protein. This enzyme has two known catalytic activities, DNA-dependent DNA polymerase and a 3' → 5'-exonuclease activity that is active on both single-stranded and double-stranded DNA. Both of these activities are also present in purified gene 5 protein alone, although the DNA polymerase activity and the double-stranded exonuclease are both greatly increased by the binding of thioredoxin. This increase has been attributed to the high processivity conferred by the thioredoxin subunit. Modified T7 DNA polymerase is able to incorporate the nucleotide analogs used for DNA sequencing. The most commonly used method for sequencing with modified T7 DNA polymerase was introduced by Tabor and Richardson to take advantage of some of its useful properties. The results of a sequencing experiment run on a high-resolution denaturing electrophoresis gel are also discussed in this chapter.