Analysis of single nucleotide incorporation reactions by capillary electrophoresis.
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.
Hanes
Jeremiah W
JW
Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX 78712, USA.
Johnson
Kenneth A
KA
D001896:Boron Compounds; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D017931:DNA Primers; D009711:Nucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase