Sizing Picograms of DNA by Single-Molecule Counting

Dmitry Torchinsky, Chemical Physics, Tel Aviv University, Tel Aviv, Israel

Some biological samples provide only few tens of nanograms of DNA, while requiring high resolution, quantitative sizing before further manipulation. We present a technique for sizing pictogram quantities of DNA by single-molecule analysis with fluorescence microscope.

An unknown DNA sample is added with a reference sample containing two DNA populations of known length labeled with CY5 fluorescent dye and the mixture is introduced with YOYO-1 fluorescent intercalating dye. The staining of the YOYO-1 molecules is homogenous, resulting in proportionality between molecule’s intensity and its length.

To image the sample it is stretched on positively activated glass coverslips and imaged in an epi-fluorescence microscope in two channels, corresponding to the excitation lights of the fluorophores. By overlaying the two channels the reference molecules are separated from the rest and their intensities are used to create a calibration curve for conversion of intensity to length.

We show that this method provides relatively easy, fast and cheap way for quantitative sizing of pictogram amounts of DNA with high resolution and sensitivity. Moreover, the use of an autofocus feature for automatic data acquisition and a program for image analysis for automatic data extraction allows us to analyze hundreds of thousands of molecules providing strong statistical basis for our results.