Nuclease P1 digestion/high-performance liquid chromatography, a practical method for DNA quantitation

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Accurate DNA quantification is crucial for molecular biology applications.
• Existing methods like UV absorbance and dye binding can be unreliable due to impurities.
• A need exists for a robust and practical DNA quantification method.

Purpose of the Study:

• To develop and validate a practical and accurate method for DNA quantification.
• To compare the performance of the new method against established techniques.

Main Methods:

• Developed a "nuclease P1/HPLC" method using a single enzyme for DNA digestion to nucleotides.
• Quantified resulting 2 -deoxynucleoside 5 -monophosphates via High-Performance Liquid Chromatography (HPLC) with UV detection.
• Applied the method to various genomic DNA samples (calf thymus, pig liver, mouse skin) with different purification histories.

Main Results:

• The nuclease P1/HPLC method demonstrated high agreement (within 10%) with the reference acid hydrolysis/HPLC method for DNA amounts in the 19-54 microg range.
• UV absorbance and dye-binding assays showed significant deviations (up to 30-40%) compared to the reference and nuclease P1/HPLC methods.
• Normalized DNA concentrations showed nuclease P1/HPLC values (1.01±0.07) closely matching the reference, while dye binding (0.8±0.17) and UV (1.1±0.1) diverged.

Conclusions:

• The nuclease P1/HPLC method is a practical and accurate approach for DNA quantification.
• It is recommended over UV absorbance and dye-binding assays when precise DNA quantification is essential, especially with potentially impure samples.
• The method's reliability is demonstrated across different DNA sources and purification methods.