Analysis of single nucleotide incorporation reactions by capillary electrophoresis.

Abstract:

Single nucleotide incorporation assays have been used to probe the kinetic parameters of many DNA and RNA polymerases. Traditionally, oligonucleotide primers are 5'-(32)P labeled using T4 kinase and annealed to a complementary template with a 5' overhang. To quantify the reaction kinetics, the products of the primer extension reactions are usually separated using denaturing polyacrylamide gel electrophoresis and quantified using a phosphorimager or other method to measure radioactivity. We have developed a method using a 5' fluorescently labeled oligonucleotide to examine the kinetics of single nucleotide incorporation catalyzed by recombinant human mitochondrial polymerase gamma (Pol gamma) holoenzyme. Using laser-induced fluorescence detection in the P/ACE MDQ instrument, primers 5' labeled with fluorescent probes such as 6-carboxyfluorescein can be rapidly separated and quantified. However, we also show that only select probes can be used, presumably due to unfavorable interactions between Pol gamma and certain 5' labels.

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