POLYNUCLEOTIDE HIGH PERFORMANCE AFFINITY CHROMATOGRAPHY

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The proposed experiments will develop a powerful method for polynucleotide (PN) separations, High Performance Affinity Chromatography (PN-HPAC). PN-HPAC may provide the means for rapid, clinical diagnosis for inherited genetic disease where the lesion on DNA is known. Molecular biology, and its applications to human health and well-being should also benefit from the development of PN-HPAC. The method involves attaching a defined DNA sequence to macroporous silica supports and preparing a chromatography column. The attached DNA will specifically hybridize by base pairing with complementary PN strands, even in the presence of other PN sequences. The specificity of PN hybridization should allow highly selective chromatography; by analogy, PN-HPAC represents a sort of "reversed Southern hybridization". Calculations presented suggest that small columns (1 x 10 mm) with kinetics should allow rapid separations to be performed. Preparatively, the columns will be used to rapidly separate mRNA, plasmids, and phages from crude cellular extracts. Analytically, the columns should be useful both clinically and for research; model studies testing the selectivity of PN-HPAC will demonstrate the feasibility of these applications. Other experiments and the susceptibility of isolated M13 phage PN- HPAC in recombinant DNA experiments and transfection of E. coli will be established. These model studies should establish PN-HPAC as a valuable tool for molecular biology.