DNA Quantification in Nanoliter Volumes

Abstract.

In recent years high throughput screening (HTS) methods have become an essential tool in discovery programs within the analytical and pharmaceutical industry. In addition, genomics and proteomics have flourished as a result of the advancements made in terms of miniaturization and parallelization of fluorescence assays, this allowing for a decrease of the consumption of expensive materials and leading to faster times of analysis. Sample volumes can be drastically reduced (down to 75 nL per cavity) compared to assays performed in microtiter plates by making use of nanotiter plates. The article describes the development of a miniaturized exonuclease-based assay that can be used for quantitative determination of non-labeled DNA. The assay utilizes fluorescence resonance energy transfer (FRET) and is carried out in a homogeneous format, with detection in the form of a direct readout of the binding and exonuclease event. In this method a fluorescence labeled FRET probe is incubated for hybridization with the non-labeled sample DNA. Exonuclease is added and cuts the double stranded specifically. The FRET is interrupted and the increasing fluorescence can be analyzed directly without separation or purification. The homogeneous DNA assay uses only commercially available materials and the entire assay takes less than 15 min. By using a FRET probe consisting of a DNA strand labeled on one end with a fluorophore and with an acceptor molecule on the other, there is no need of labeling the sample DNA. The assay enables facile differentiation between DNAs with complementary, non-complementary and single nucleotide polymorphism sequences, respectively.