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Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy
Published on: July 18, 2011
Visualization of alkali-denatured supercoiled plasmid DNA by atomic force microscopy
Jia Yu1, Zhenfeng Zhang, Kou Cao
1Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, PR China.
Biochemical and Biophysical Research Communications
|July 8, 2008
Summary
Alkali denaturation of supercoiled DNA occurs within a narrow pH range, altering its structure and migration patterns. This process results in a stable, conformationally changed DNA with kinks and shorter contour lengths.
Area of Science:
- Molecular Biology
- Biochemistry
- Biophysics
Background:
- Supercoiled DNA plays crucial roles in cellular processes.
- Understanding DNA structural transitions is vital for molecular biology.
Purpose of the Study:
- To investigate the effects of alkali denaturation on supercoiled plasmid DNA.
- To characterize the structural changes and conformation of alkali-denatured supercoiled DNA.
Main Methods:
- Treatment of supercoiled plasmid DNA (pBR322) with varying concentrations of NaOH.
- Analysis of DNA denaturation using gel electrophoresis.
- Visualization of alkali-denatured DNA using atomic force microscopy (AFM).
Main Results:
- Alkali denaturation of supercoiled DNA occurred in a narrow pH range (12.88-12.90).
- Denatured DNA migrated faster than supercoiled DNA on gel electrophoresis.
- AFM revealed rough surfaces, kinks, bulges, and inhomogeneous diameters in denatured DNA.
- Apparent contour lengths decreased by 16% (pBR322, pACYC184) and 50% (pJGX15A).
Conclusions:
- Alkali-denatured supercoiled DNA adopts a stable conformation.
- The denaturation process leads to topologically constrained double strands and intrastrand secondary structures.
