SIZING FEMTOGRAM AMOUNTS OF DSDNA BY SINGLE-MOLECULE COUNTING

In this work we utilize single-molecule imaging with in-situ size calibration to accurately analyze the size and mass distribution of a given sample. We use synthetic reference DNA standards as an internal size calibrator. The reference consists of two distinct DNA populations which are synthesized and covalently labeled with Cy5 by PCR. Spiking the investigated sample with the reference standard and staining the two together with an intercalating dye cancels out the factor of variable labeling conditions. Moreover, in order to be able to size samples of unknown concentration we adjusted the labeling conditions for homogeneous staining even at an excessive dye:bp labeling ratio. During imaging, the Cy5 label of the reference DNA molecules allows their separation from the investigated sample during data analysis. The intensities of the reference molecules are used to create a calibration curve for conversion of length to intensity, which allows sizing of the unknown DNA. Our approach may find useful applications for genetic analysis where the exact size distribution of DNA molecules is critical. Examples include characterizing free circulating DNA in plasma or urine, analyzing telomeres and mitochondrial DNA, or in combination with sequencing library preparation.