Engineering of hybrid enzymes for DNA technologies

DESCRIPTION (provided by applicant): The main objective of this SBIR Phase I application is to develop a generic approach for construction by methods of gene engineering hybrid DNA metabolizing enzymes with enhanced processivity. Particularly, a set of processive thermostable DNA polymerases, capable of extensive rapid replication, efficient recycling between substrate molecules and suitable for both LA-PCR and most challenging DNA sequencing applications will be developed. A number of plasmid constructs will be made and the encoding chimeric proteins will be expressed in E. coli, purified and assayed for enhanced processivity, i.e for capability to extend a primer for 1 kb or more prior to dissociating from a template, and for significantly higher affinity to a primer-template comparing to current commercial thermostable polymerases. Such improvement in basic enzyme properties will enhance both molecular biology research at universities and boost industrial genome research as well. The availability of a specialized enzyme for long PCR and advanced genomic sequencing will enhance diagnostic procedures for cancer, genetic defects and pathogens.