Monitoring of single nicks in duplex DNA by gel electrophoretic mobility-shift assay
We demonstrate that the gel electrophoretic mobility‐shift assay (EMSA) can be used for site‐selective and quantitative monitoring of nicks in linear double‐stranded DNA (dsDNA) thus allowing to expediently follow the nicking activity of enzymes or other agents targeted to a designated dsDNA site. A...
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Published in | Electrophoresis Vol. 23; no. 15; pp. 2384 - 2387 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
Weinheim
WILEY-VCH Verlag
01.08.2002
WILEY‐VCH Verlag |
Subjects | |
Online Access | Get full text |
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Summary: | We demonstrate that the gel electrophoretic mobility‐shift assay (EMSA) can be used for site‐selective and quantitative monitoring of nicks in linear double‐stranded DNA (dsDNA) thus allowing to expediently follow the nicking activity of enzymes or other agents targeted to a designated dsDNA site. At elevated temperature and/or in the presence of urea, DNA fragments carrying a single nick produced by the nicking enzyme N.BstNBI exhibit a well‐detectable gel retardation effect. On the basis of permutation analysis, the decreased electrophoretic mobility of nicked dsDNA fragments is attributed to a bend (or hinge) in the DNA double helix sequence‐specifically generated by a nick. Since nick‐induced DNA bending depends on interaction between base pairs adjacent to a nick, the change in mobility is different for nicked DNA sites with different sequences. Therefore, EMSA monitoring of differential mobility change caused by nicks within various DNA sequences could be useful for studying the differential base stacking and nearest‐neighbor energetics. |
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Bibliography: | ArticleID:ELPS2384 ark:/67375/WNG-2S6DNN02-6 istex:701B9D83964647E839FD02F9ECD4213B6CABFA00 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0173-0835 1522-2683 |
DOI: | 10.1002/1522-2683(200208)23:15<2384::AID-ELPS2384>3.0.CO;2-O |