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
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
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چکیده انگلیسی
Single nucleotide incorporation assays have been used to probe the kinetic parameters of many DNA and RNA polymerases. Traditionally, oligonucleotide primers are 5â²-32P labeled using T4 kinase and annealed to a complementary template with a 5â² 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 5â² 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 5â² 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 5â² labels.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 340, Issue 1, 1 May 2005, Pages 35-40
Journal: Analytical Biochemistry - Volume 340, Issue 1, 1 May 2005, Pages 35-40
نویسندگان
Jeremiah W. Hanes, Kenneth A. Johnson,