Sizing of large DNA molecules by hook formation in a loose matrix

We present details on how to implement a newly developed methodology, Optical Contour Maximization (OCM), for accurately sizing large DNA molecules. Agarose gel containing stained DNA is cast between a slide and a coverslip, and molecules moved by an applied electrical field are observed using fluorescence microscopy. The molecules of interest lie in the interface formed between the coverslip and gel. DNA movement in this region is largely confined to lateral motions. When a DNA molecule snags an obstacle, it elongates, forming a metastable hook that can persist for several seconds. We found that the longest observed hook contour length can be determined from rapidly collected images. This maximized length shows a linear correlation with reported size [X.H. Guo, E.J. Huff & D.C. Schwartz, Nature 359, 783 (1992)] Successful measurements require a critical balance between the voltage needed for full elongation, and the unwanted effects of too large a voltage: fewer metastable hooks form, they dissipate faster, molecule breakage becomes a problem, and faster image collection becomes necessary. We measured apparent contour length as a function of applied voltage and determined an optimal voltage for our apparatus, using a singly anchored 114kb molecule. The measurement precision is estimated from the distribution of results. We expect that OCM will find utility in physical mapping and molecular karyotyping of lower eucaryotes of medical importance.