There are numerous methods suitable for quantifying the number of nucleic acids in a sample, but UV-Vis absorbance is the primary method for determining a sample’s concentration and purity of DNA or RNA.
Both single- and double-stranded DNA strongly absorb ultraviolet light with a peak absorbance wavelength of 260nm. Simple concentration and purity measurements involve a spectrum of light being shone through a sample either directly in microvolume methods or contained within a plastic or glass cuvette. According to the Beer-Lambert law, the attenuation of light through the sample is proportional to the concentration and the distance the light travels through the sample
However, DNA is not the only substance of interest that absorbs light on the sub-visible spectrum. The aromatic amino acids of various proteins and ribonucleic acid (RNA) both absorb light at roughly 260 – 280nm. Given the difficulty in ensuring absolute sample purity, samples may contain various contaminants that could contribute to UV absorbance. This could cause an overestimation of the concentration of DNA in the sample. Purity ratios that assess the contribution of these contaminants to measurements are used to identify potential sample issues.
How UV-Vis Spectrophotometry Works
A UV-Vis spectrophotometer is equipped with one or more channels of an incident light source that covers the full ultraviolet-visible (UV-Vis) electromagnetic spectrum (190 – 840nm). Analysts selectively interrogate the isolated sample with pulses of light across specific wavelength ranges. The UV-Vis optical detector acquires the transmission spectra of samples in real time, obtaining high-resolution measurements of the sample’s total spectral output. This resolution and measurement accuracy is critical for quantitating analytes of interest based on their unique spectral fingerprints.
UV-Vis Quantitation of DNA with DeNovix
DeNovix is a leading authority in the field of UV-Vis spectrophotometry. The management team has been intrinsically involved in the development of microvolume technologies, and their CEO was previously a founding partner and president of NanoDrop Technologies Inc. DeNovix has since developed one of the world’s leading UV-Vis spectrophotometers, performing with unmatched precision and the widest dynamic range on the market.
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