Describe an experimental approach to determining the processivity of a DNA polymerase (i.e., the number of nucleotides incorporated per chain per polymerase binding event).
Describe an experimental approach to determining the processivity of a polymerase (i.e., the number of nucleotides incorporated per chain per polymerase binding event).
Use mixture of labeled forward primer and run four polymerase reactions in DNA template for each terminating dNTP. Stop reactions after a brief incubation period. After reaction determine the molecular weight of the labeled DNA by gel electrophoresis and autoradiography. As a Singer sequencing method it will give information about sequence of template and the maximum number of nucleotides added per chain during the incubation, and this gives the processivity (number of molecules incorporated per polymerase-DNA encounter).
Use a 5′ end-labeled primer and run a polymerase reaction in DNA template excess, so that any polymerase molecule that dissociates will bind to a new DNA strand that has not been copied; hence, every DNA strand that is copied will have been copied in only one encounter with a polymerase molecule. Stop reactions after a brief incubation period then determine the molecular weight of the labeled DNA by gel electrophoresis and autoradiography. The molecular weight will give the number of nucleotides added per chain during the incubation, and this gives the processivity (number of molecules incorporated per polymerase-DNA encounter).
Use DNA template with known sequence, label one primer, used in mixture one labeled dNTP. So every new copied DNA would have target (label) for separation them from template strand. Stop reactions after a brief incubation period then prepare single stranded DNA library and disunite strands that includes labeled primer. Determine the molecular weight of the labeled DNA by gel electrophoresis and autoradiography. The intensity of the glow and knowledge about positions of labeled base in the sequence will give the number of nucleotides added per chain during the incubation, and this gives the processivity (number of molecules incorporated per polymerase-DNA encounter).