[en] Gel retardation analysis, or band shift assay, is technically the simplest method to investigate protein-nucleic acid interactions. In this report, we describe a nonradioactive band shift assay using a fluorescent DNA target and an ALFexpress automatic DNA sequencer in place of the current method that utilizes radioactively end-labeled DNA target and a standard electrophoresis unit. In our study, the dsDNA targets were obtained by annealing two synthetic oligonucleotides or by PCR. In both cases, a molecule of indodicarbocyanine (CY5) was attached at the 5' OH end of one of the two synthetic oligonucleotides, with a ratio of one molecule of fluorescent dye per molecule of dsDNA. To demonstrate the feasibility of this new band shift assay method, the DNA-binding proteins selected as models were the BlaI and AmpR repressors, which are involved in the induction of the Bacillus licheniformis 749/I and Citrobacter freundii beta-lactamases, respectively. The results show that the use of an automatic DNA sequencer allows easy gel retardation analysis and provides a fast, sensitive, and quantitative method. The ALFexpress DNA sequencer has the same limit of detection as a laser fluorescence scanner and can be used instead of a FluorImager or a Molecular Imager.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Filée, Patrice ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Delmarcelle, Michaël ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Thamm, Iris ; Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
Joris, Bernard ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Language :
English
Title :
Use of an Alfexpress DNA Sequencer to Analyze Protein-Nucleic Acid Interactions by Band Shift Assay
Publication date :
May 2001
Journal title :
BioTechniques
ISSN :
0736-6205
eISSN :
1940-9818
Publisher :
Eaton Publishing Co., United States - Massachusetts
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Bibliography
Bartowsky, E. and S. Normark. 1993. Interactions of wild-type and mutant AmpR of Citrobacter freundii with its target DNA. Mol. Microbiol. 10:555-565.
Dubnau, D.A. and M.R. Pollock. 1965. The genetics of the Bacillus licheniformis penicillinase: a preliminary analysis from studies on mutation and inter-strain and intra-strain transformations. J. Gen. Microbiol. 41:7-21.
Galas, D.J. and A. Schmitz. 1978. DNase footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 9:3157-3170.
Imanaka, T., M. Nakae, T. Ahta, and M. Takagi. 1992. Design of a temperature-sensitive penicillinase repressor by replacement of proline in predicted bet-turn structures. J. Bacteriol. 174:1423-1425.
Jacobs, C., J.M. Frère, and S. Normark. 1997. Cytosolic intermediates for cell wall biosynthesis and degradation control inducible β-lactam resistance in gram-negative bacteria. Cell 88:823-832.
Kobayashi, T., Y.F. Zhu, N. Nicholls, and J.O. Lampen. 1987. A second regulatory gene blaRI, encoding a potential penicillin-binding protein required for induction of β-lactamase in Bacillus licheniformis. J. Bacteriol. 169:3873-3878.
Lane, D., P. Prentki, and M. Chandler. 1992. Use of gel retardation to analyze protein-nucleic acid interactions. Microbiol. Rev. 56:509-528.
Wittman, V., H.C. Lin, and A.C. Wong. 1993. Functional domains of the penicillinase repressor of Bacillus licheniformis. J. Bacteriol. 175:7383-7390.
Wittman, V. and H.C. Wong. 1988. Regulation of the penicillinase genes of Bacillus licheniformis: interaction of the pen repressor with its repressors. J. Bacteriol. 170:3206-3212.
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