Improved quantitation of DNA curvature using ligation ladders.
It is often desirable to estimate accurately the local shape of DNA molecules. Such measurements are useful in understanding the intrinsic contribution of DNA sequence to curvature, as well as in assessing the effects of chemical modifications. We have been investigating the effects of asymmetric phosphate neutralization on DNA shape using the well-characterized ligation ladder approach developed by Crothers and co-workers [D.M. Crothers and J.Drak (1992) Meth. Enzymol.,212, 46-71]. This technique is remarkably sensitive to differences in DNA shape. We now report a general quantitative assay of DNA curvature that we have validated using a set of phased A(5)tract standards. This approach allows simultaneous estimation of helix axis deflection magnitude and direction when a test sequence is monitored in at least three phasings relative to a reference A(5-6)tract in short DNA duplexes. Analysis using this improved approach confirms our published data on DNA curvature due to electrostatic effects.
Ross
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Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN 55905, USA.
Den
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Hardwidge
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Maher
L J
LJ
D000107:Acetylation; D001483:Base Sequence; D002138:Calibration; D002412:Cations / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011088:DNA Ligases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016018:Least-Squares Analysis; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism; D015203:Reproducibility of Results; D055672:Static Electricity
D002412:Cations; D009838:Oligodeoxyribonucleotides; D010710:Phosphates; D011743:Pyrimidines; D004247:DNA; D011088:DNA Ligases