Fractionation of DNA on hydroxyapatite with a base-specific complexing agent

Area of Science:

• Biochemistry
• Molecular Biology
• Chromatography

Background:

• Separating DNA mixtures based on G+C content is crucial for genomic analysis.
• Existing methods like CsCl density gradients have limitations in capacity and resolution.

Purpose of the Study:

• To develop a novel chromatographic technique for efficient DNA separation by G+C content.
• To utilize a G.C-specific DNA ligand for enhanced separation resolution and capacity.

Main Methods:

• A high-capacity hydroxyapatite column was prepared.
• A G.C-specific DNA ligand, 2-methyl-3-amino-7-dimethylamino-5-phenyl-phenazinium cation (PNR), was employed.
• DNA mixtures were separated based on differential binding to the ligand on the hydroxyapatite column.

Main Results:

• The method effectively separates DNA molecules based on G+C content, with G.C-rich DNA eluting earlier.
• The resolution approaches that of CsCl density gradients, but with significantly larger column capacity.
• Separation is independent of DNA molecular weight, allowing for G+C-based fractionation of mixed-size samples.
• Demonstrated successful separation of G.C-rich minor components from calf thymus DNA.

Conclusions:

• This PNR-ligand-based hydroxyapatite chromatography is a powerful tool for DNA G+C content separation.
• The technique offers advantages in capacity, resolution, and molecular weight independence over traditional methods.
• It is particularly useful for analyzing G.C-rich DNA fractions from complex eukaryotic genomes.