کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
10533808 961894 2005 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Analysis of single nucleotide incorporation reactions by capillary electrophoresis
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Analysis of single nucleotide incorporation reactions by capillary electrophoresis
چکیده انگلیسی
Single nucleotide incorporation assays have been used to probe the kinetic parameters of many DNA and RNA polymerases. Traditionally, oligonucleotide primers are 5′-32P 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 γ (Pol γ) 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 γ and certain 5′ labels.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 340, Issue 1, 1 May 2005, Pages 35-40
نویسندگان
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