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Comparison of three common DNA concentration measurement methods
Xiaofei Li1, Yuhua Wu1, Li Zhang1
1Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, People's Republic of China.
Analytical Biochemistry
|February 6, 2014
Summary
Comparing DNA quantification methods, the diphenylamine reaction showed highest accuracy with contaminants. PicoGreen dye is best for degraded DNA, while UV absorption effectively detects impurities in DNA solutions.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Accurate DNA quantification is crucial for various molecular biology applications.
- Established methods include UV absorption, fluorescence staining (PicoGreen dye), and the diphenylamine reaction.
- The optimal method for quality assurance across different sample types remains unclear.
Purpose of the Study:
- To comprehensively compare the accuracy and reliability of UV absorption, PicoGreen dye, and diphenylamine reaction methods for DNA concentration measurement.
- To evaluate the performance of these methods under varying conditions, including DNA purity, degradation, and the presence of common contaminants.
Main Methods:
- Comparison of three distinct DNA quantification techniques: UV absorption, PicoGreen dye fluorescence, and diphenylamine colorimetric assay.
- Assessment of method accuracy using high-purity DNA standards.
- Evaluation of method sensitivity to DNA degradation via enzymatic digestion.
- Analysis of method robustness against common contaminants such as transfer RNA (tRNA), proteins, and organic chemicals.
Main Results:
- All three methods accurately quantified high-purity DNA solutions.
- PicoGreen dye method showed high sensitivity to DNA degradation.
- The diphenylamine reaction method exhibited the highest accuracy in the presence of contaminants (approx. 10% error).
- UV absorption was susceptible to all tested impurities, while PicoGreen was affected by tRNA and protein.
Conclusions:
- The diphenylamine reaction method is most accurate for DNA quantification in contaminated samples.
- The PicoGreen dye method is suitable for degraded DNA or DNA from processed sources.
- UV absorbance is best suited for assessing the impurity levels within DNA solutions.
