An Engineered RNA/DNA Polymerase to Increase Speed and Economy of DNA Sequencing

41294 November 19, 1996 Promega Corporation DNA sequence information is the cornerstone for considerable experimental design and analysis in the biological sciences, but it is time consuming and expensive. The project will focus on advancing DNA sequencing and reducing its cost by creating a new enzyme that eliminates the need for an oligonucleotide primer to initiate DNA synthesis at a defined site, and that can use dideoxy nucleotides for chain termination. The new method should reduce the time and cost required to obtain DNA sequences, and enhance the speed and cost effectiveness of current DNA sequencing technologies. Phase I will purify mutant polymerases, develop protocols for rapid small scale mutant enzyme purification, evaluate the purified mutants for properties relevant to DNA sequencing, develop facile mutagenesis schemes, and produce mutant RNA/DNA polymerases with altered promoter recognition. Phase II will refine properties of the mutant by: (1) expanding the number of mutations examined using the purification protocols, assays, and mutagenesis screening methods developed in Phase I, (2) examining the effect of each mutation on enzymatic properties important to DNA sequencing applications, and (3) optimizing conditions for sequencing performance. Commercial Applications and Other Benefits as described by the awardee: Anticipated products include systems for direct sequencing of plasmid and phage DNA without the use of oligonucleotide primers to initiate synthesis. These reagent systems should be easily incorporated into high throughput, automated DNA sequence analysis systems, and should be compatible with other sequencing technologies and with existing commercial cloning vectors and PCR fragments used for sequencing applications. Other commercially useful properties may include the ability to incorporate modified nucleotides for non-isotopic detection of nucleic acids, and the facile production of ssDNA.