Design and calibration of a semi-synthetic DNA phasing assay.
Electrophoretic assays of intrinsic DNA shape and shape changes induced by ligand binding are extremely useful because of their convenience and simplicity. The development of calibrations and empirical quantitative relationships permits highly accurate measurement of DNA shape using electrophoresis. Many conventional analyses employ the unidirectional ligation of short DNA duplexes. However, many oligonucleotides (typically more than 20) must often be synthesized for a single experiment. Additionally, the length of the DNA duplex can become limiting, preventing the analysis of certain DNA sequences. We now describe a semi-synthetic electrophoretic phasing method that offers several advantages, including a reduced number of required synthetic oligonucleotides, the ability to analyze longer DNA duplexes and a simplified approach for data analysis. We characterize semi-synthetic DNA probes in electrophoretic phasing assays by ligation of synthetic duplexes containing A(5) tracts between two longer restriction fragments. Upon electrophoresis, the gel mobility is strongly correlated with the predicted DNA curvature provided by the reference A(5) tracts. Having obtained this calibration, we show that the semi-synthetic phasing assay can be readily and economically applied to analyze DNA curvature induced by DNA charge modifications and DNA bending due to peptide binding.
Hardwidge
P R
PR
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN 55905, USA.
Zimmerman
J M
JM
Maher
L J
LJ
D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D004586:Electrophoresis / Q000379:methods; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D011494:Protein Kinases