Estimating the Concentration of DNA by Fluorometry Using Hoechst 33258

Area of Science:

• Molecular Biology
• Biochemistry

Background:

• Spectrophotometry is a common method for DNA quantification.
• Fluorometry offers increased sensitivity for detecting low DNA concentrations.

Purpose of the Study:

• To describe a fluorometric assay for DNA concentration measurement using Hoechst 33258.
• To compare the sensitivity of fluorometry to spectrophotometry for DNA quantification.

Main Methods:

• DNA samples of known and unknown concentrations were incubated with the fluorochrome Hoechst 33258.
• Absorption values were measured and compared to a standard dilution series.
• Concentration of unknown samples was determined by interpolation.

Main Results:

• Fluorometry allows detection of nanogram quantities of DNA, surpassing spectrophotometry sensitivity.
• The assay is effective for DNA molecules exceeding approximately 1 kilobase (kb).
• Hoechst 33258 exhibits poor binding to DNA fragments smaller than 1 kb.

Conclusions:

• Fluorometry with Hoechst 33258 is a sensitive method for quantifying DNA concentrations.
• This assay is suitable for DNA samples larger than 1 kb.
• The sensitivity of this method enables detection of very low DNA amounts.