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Abstract

A new approach in mapping restriction fragments by means of primed extension was proposed but was found to be unfeasible after studying the extent of T4 polymerase mediated DNA synthesis. The maximum length of DNA replication mediated by T4 polymerase was studied using ØX-174 DNA as template primed by a restriction fragment of the same DNA. Both nucleotide incorporation kinetics and alkaline gel electrophoresis were used to study the products of DNA synthesis. Although the incorporation kinetics suggested that the primer was extended by approximately 100 nucleotides, the electrophoretic mobilities of the products suggested much less extension. The effect of T4 gene 32 protein (unwinding protein) was also studied. This protein was purified by DNA cellulose chromatography to near homogeneity and was shown to be nuclease free. The purified protein stimulated nucleotide incorporation three-fold when added to the usual T4 polymerase reaction mixture. Contrary to the kinetic results, however, the gel mobilities of the products again showed only limited extension of the primer.