Literature Seminar

The most commonly used method of quantification for single- and double-stranded nucleic acid species is UV spectrophotometry. Beer’s law relates the absorbance of a sample with the pathlength, concentration and attenuation coefficient (ε). For nucleic acids, an average mass attenuation coefficient is applied to approximately quantify the species independent of base composition. There are two main issues that need to be considered when quantifying nucleic acids with spectrophotometry: hypochromicity in double-stranded species, and absorbance of contaminants at the incident wavelength. The hypochromicity phenomenon explains that double-stranded nucleic acids absorb less light than their corresponding single-strands due to base stacking interactions. Additionally, compounds like phenol are used in DNA/RNA extraction, and a phenol contamination can significantly change the absorbance at the incident 260 nm wavelength. As a means of improving the accuracy of quantification by UV spectrophotometry, Nwokeoji et al proposed a denaturing step in 50% DMSO to eliminate the impact of hypochromicity on absorbance measurements. This technique is especially useful for quantifying double-stranded RNA, as traditional methods of denaturing RNA can result in hydrolysis. This talk will focus on previous work that seeks to quantify RNA and DNA as well as the newly proposed methods of RNA and DNA sample preparation that allow for a higher level of accuracy to be achieved when quantifying with spectrophotometry.