کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1176870 961895 2011 9 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Direct and continuous fluorescence-based measurements of Pyrococcus horikoshii DNA N-6 adenine methyltransferase activity
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Direct and continuous fluorescence-based measurements of Pyrococcus horikoshii DNA N-6 adenine methyltransferase activity
چکیده انگلیسی

N-6 methylation of adenine destabilises duplex DNA and this can increase the proportion of DNA that dissociates into single strands. We have investigated utilising this property to measure the DNA adenine methyltransferase-catalyzed conversion of hemimethylated to fully methylated DNA through a simple, direct, fluorescence-based assay. The effects of methylation on the kinetics and thermodynamics of hybridisation were measured by comparing a fully methylated oligonucleotide product and a hemimethylated oligonucleotide substrate using a 13-bp duplex labeled on adjacent strands with a fluorophore (fluorescein) and quencher (dabcyl). Enzymatic methylation of the hemimethylated GATC site resulted in destabilisation of the duplex, increasing the proportion of dissociated DNA, and producing an observable increase in fluorescence. The assay provides a direct measurement of methylation rate in real time and is highly reproducible, with a coefficient of variance over 48 independent measurements of 3.6%. DNA methylation rates can be measured as low as 3.55 ± 1.84 fmol s−1 in a 96-well plate format, and the assay has been used to kinetically characterise the Pyrococcus horikoshii DNA adenine methyltransferase.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 418, Issue 2, 15 November 2011, Pages 204–212
نویسندگان
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