High Throughput Screen for Novel DNA Polymerases
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AbstractDESCRIPTION (provided by applicant): Thermos table DNA polymerases are indispensable reagents in two of the most commonly used methods in health research today, DNA amplification and sequencing, as well as in SNP detection. While Taq DNA polymerase and other available enzymes are certainly adequate for many applications, they all have activities that will compromise certain results. For example, genetic analysis of the small nucleotide repeat sequences associated with a number of human diseases is compromised by the PCR stutter and slippage induced expansion artifacts generated by Taq. Discovery of new thermos table DNA polymerases has traditionally been a time and resource-intensive activity. The purpose of this work is to create a high throughput system for identifying new thermos table bacterial and archaeal DNA polymerases. The aim of this work is not to identify all possible DNA polymerases, but to rapidly identify new and valuable DNA polymerases that are both not toxic to E. coli, and are stably expressed in E. coli. In E. coli, either DNA polymerase I, the product of the polA gene, or recombinase A, the product of the recA gene, is required for growth of E. coli. Temperature sensitive mutants in the polA gene, when combined with a constitutive recA mutation, verify this; these mutants grow at low, but not high temperature. These temperature sensitive mutants are not suitable for high throughput screening of DNA libraries for a number of reasons, primarily the high background of false positives generated by reversion to the wild type. We propose to engineer a polA gene in E. coli with a pair of on-off switches, one at each end of the gene, that are controlled by chemical inducers. After transformation with foreign DNA, the new polA gene is switched off, and only those clones containing an expressed foreign DNA polymerase will be able to grow. This will allow rapid identification of new, stably expressed, active DNA polymerase genes. It is expected that many of these new DNA polymerases will be useful in research furthering our understanding of the genetic bases of diseases.
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