Assessing Nucleic Acid Purity Via Absorption Spectroscopy
The interaction of nucleic acids with light provides a useful tool to not only quantify the amount of DNA or RNA present in a solution but also to assess its potential contamination by proteins and other biomolecules. As the concentration and purity of DNA and RNA are critical for downstream applications, establishing these values early on can reduce or eliminate errors, save time, and ensure optimum results by identifying problematic samples before they are used.
What is Absorption Spectroscopy and How does it work?
Absorption spectroscopy relies on the fact that molecules interact with wavelengths of light differently depending on their chemical structures. For nucleic acids, the nitrogenous bases interact most strongly with light, strongly absorbing 260 nm photons of light. For proteins, the conjugated rings of the amino acids phenylalanine, tyrosine, and tryptophan interact the most with light, absorbing photons at 280 nm.
To measure the concentration of DNA/RNA in a pure sample, the sample is placed inside a cuvette which is placed inside a spectrophotometer. This machine exposes the sample to a known amount of a known wavelength of light – 260 nm - and detects the light that is transmitted through the sample and reaches a photodetector. A sample with higher DNA/RNA concentration will absorb more light, producing a higher optical density(OD). The same principle is true for proteins, but the wavelength used will be 280 nm.
The Beer-Lambert Law relates the concentration of the absorbing molecule to the amount of absorbed light:
A = log10 Io/I = elc
where: A = Absorbance
Io = Initial Intensity of light
I = intensity of light measured at photodetector
e = molar absorption coefficient of the analyte. For double-stranded DNA at 260 nm, e = 0.020 (μg/ml)−1 cm−1. For single-stranded RNA at 260 nm, e = 0.025 (μg/ml)−1 cm−1. For short single-stranded oligonucleotides, e depends on length and base composition.
l = Optical path length of the cuvette
c = Molar concentration of analyte in solution
Assessing Purity
While absorption spectroscopy is useful for quantifying analytes in solution, it can also be used to assess sample purity – if the contaminants show unique absorption signatures. In nucleic acid purification procedures, the most common contaminants are proteins and residual components of the kit - alcohols, phenols, or salts. To assess the extent of protein contamination in a nucleic acid preparation, absorbance readings at both 260 nm and 280 nm must be taken. This is because DNA/RNA absorbs at 260 nm while protein absorbs at 280.nm Next, the A260/A280 ratio is calculated. The A260/A280 ratio for pure RNA is 2.0, and the ratio for pure DNA is 1.8. Lower values may indicate a low yield of nucleic acid or contamination by protein, phenols, or other chemicals which absorb strongly at 280 nm. For samples showing contamination, further processing via DNA cleanup protocols can remove the contaminants, rescuing precious samples.
Addressing Limitations
Absorption spectroscopy is an invaluable technique for nucleic acid quantification and purity assessment due to its speed and ease of use, it has limitations. To begin with, absorption spectroscopy relies on bulk measurement. It provides the overall absorbance at a specific wavelength but does not distinguish between different types of nucleic acids. Additional methods, such as gel electrophoresis or sequencing, may be required for additional characterization. Second, absorption spectroscopy assumes the sample contains only the analytes of interest and if free from interfering substances. Careful sample preparation is necessary to minimize interferences and protect the integrity of results. Third, while spectroscopy can be a highly sensitive technique, detecting low concentrations of nucleic acids and proteins can be difficult. The sensitivity of the technique is determined by the instrument used, the path length of the cuvette, and the extinction coefficients of the analytes. For accurate quantification of low-abundance samples, alternative methods with higher sensitivity – such as fluorescence or PCR-based techniques – may be required. Lastly, measuring nucleic acids and proteins via absorption spectroscopy involves subjecting these biomacromolecules to high-energy UV light. This exposure can lead to molecular damage. For samples that must be used to downstream applications, care must be taken to optimize exposure times to minimize potential for sample damage.
Nucleic Acid Purification – https://www.gbiosciences.com/Molecular-Biology/GET-Plasmid-DNA-Miniprep
DNA cleanup - https://www.gbiosciences.com/Molecular-Biology/DNA-Clean-Up-Concentration/GET-CLEAN-DNA
Handbook: Molecular Biology
