Microvolume quantitation of nucleic acids

Quantitation of DNA and RNA by absorbance and fluorescence spectroscopy has been a powerful tool in life sciences for decades. Classic methods of nucleic acid quantitation require the filling of devices, such as cuvettes and capillaries, with sample (traditional methodologies are described in APPENDIX 3D). Analysis of microvolume samples has become of paramount importance as more molecular biology techniques yield progressively smaller amounts of isolated sample and require accurate quantitation of nucleic acids with minimal consumption of sample. Advances in photonic technologies have resulted in a pioneering microvolume system that combines fiber optic technology with the inherent physical properties of the sample to dramatically reduce measurement volumes, removing the need for cuvettes and capillaries. Since the introduction of the first microvolume instrument, several new designs are now available, providing opportunities to measure nucleic acids using much smaller amounts of material. Altogether, these systems not only reduce measurement volume (as little as 0.5 to 2 µl), but also tend to be more efficient time-wise than traditional methods, making them useful even when sample is plentiful. The protocols in this unit are based on the most widely accepted microvolume systems and are intended as practical alternatives to traditional nucleic acid quantitation methodology.