DNA electrophoresis in uncross-linked polyacrylamide solution, studied by epifluorescence microscopy

Area of Science:

• Molecular Biology
• Biophysics
• Genetics

Background:

• Understanding DNA behavior during electrophoresis is crucial for genetic analysis and manipulation.
• Previous studies have focused on DNA migration in simple buffers or gels, with less known about its behavior in polymer solutions.

Purpose of the Study:

• To investigate the molecular conformations and migration dynamics of large human DNA fragments during electrophoresis in uncross-linked polyacrylamide solutions.
• To characterize the influence of polymer concentration and DNA size on DNA conformation and electrophoretic mobility.
• To examine the behavior of DNA-agarose complexes under similar electrophoretic conditions.

Main Methods:

• Electrophoresis of human DNA fragments (10^5 to 10^7 bases) in a polyacrylamide solution within a glass tube.
• Real-time monitoring using epifluorescence microscopy to visualize DNA conformations.
• Electrophoresis of Schizosaccharomyces pombe DNA embedded in agarose, followed by melting and enzyme treatment.

Main Results:

• In polyacrylamide, DNA adopted a 'trailing network' with a globular 'head' conformation, affecting migration velocity.
• Migration velocity correlated with the size of the 'head' and the ratio of 'head' to 'network'.
• Lambda phage DNA in buffer migrated as a globular form, consistent with macroscopic data.
• A migrating DNA-agarose complex was observed after melting, resistant to enzymatic dissociation.

Conclusions:

• Polyacrylamide solutions induce unique DNA conformations ('trailing network' and 'head') that govern electrophoretic mobility.
• The observed DNA-agarose complex exhibits unusual stability, suggesting potential applications in DNA purification or analysis.