کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1356148 981092 2008 11 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Improvement of DNA adenylation using T4 DNA ligase with a template strand and a strategically mismatched acceptor strand
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آلی
پیش نمایش صفحه اول مقاله
Improvement of DNA adenylation using T4 DNA ligase with a template strand and a strategically mismatched acceptor strand
چکیده انگلیسی

DNA with a 5′-adenylpyrophosphoryl cap (5′-adenylated DNA; AppDNA) is an activated form of DNA that is the biochemical intermediate of the reactions catalyzed by DNA ligase, RNA ligase, polynucleotide kinase, and other nucleic acid modifying enzymes. 5′-Adenylated DNA is also useful for in vitro selection experiments. Efficient preparation of 5′-adenylated DNA is therefore desirable for several biochemical applications. Here we have developed a DNA adenylation procedure that uses T4 DNA ligase and is more reliable than a previously reported approach that used the 5′-phosphorylated donor DNA substrate to be adenylated, a DNA template, and ATP but no acceptor strand. Our improved DNA adenylation procedure uses the above components as well as an acceptor strand that has a strategically chosen C–T acceptor–template mismatch directly adjacent to the adenylation site. This mismatch permits adenylation of the donor DNA substrate but largely suppresses subsequent ligation of the donor with the acceptor, as assayed on nine different DNA substrates that collectively have all four DNA nucleotides represented at each of the first two positions. The new DNA adenylation procedure is successful using either laboratory-prepared or commercial T4 DNA ligase and works well on the preparative (2 nmol) scale for all nine of the test DNA substrates.

An improved procedure for DNA adenylation is developed. The procedure uses T4 DNA ligase, ATP, a DNA template, and an acceptor strand with a strategic C–T acceptor–template mismatch adjacent to the adenylation site. This mismatch permits efficient adenylation but suppresses subsequent ligation.Figure optionsDownload as PowerPoint slide

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Bioorganic Chemistry - Volume 36, Issue 2, April 2008, Pages 46–56
نویسندگان
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