Combing of Genomic DNA from Droplets Containing Picograms of Material

Abstract

Deposition of linear DNA molecules is a critical step in many single-molecule genomic approaches including DNA mapping, fiber-FISH, and several emerging sequencing technologies. In the ideal situation, the DNA that is deposited for these experiments is absolutely linear and uniformly stretched, thereby enabling accurate distance measurements. However, this is rarely the case, and furthermore, current approaches for the capture and linearization of DNA on a surface tend to require complex surface preparation and large amounts of starting material to achieve genomic-scale mapping. This makes them technically demanding and prevents their application in emerging fields of genomics, such as single-cell based analyses. Here we describe a simple and extremely efficient approach to the deposition and linearization of genomic DNA molecules. We employ droplets containing as little as tens of picograms of material and simply drag them, using a pipet tip, over a polymer-coated coverslip. In this report we highlight one particular polymer, Zeonex, which is remarkably efficient at capturing DNA. We characterize the method of DNA capture on the Zeonex surface and find that the use of droplets greatly facilitates the efficient deposition of DNA. This is the result of a circulating flow in the droplet that maintains a high DNA concentration at the interface of the surface/solution. Overall, our approach provides an accessible route to the study of genomic structural variation from samples containing no more than a handful of cells.

Spontaneous formation of aligned DNA nanowires by capillarity-induced skin folding

“…Molecular combing and its derivatives have been used widely to obtain such nanowires using an aqueous solution of DNA molecules, where capillary forces of the solution at a receding meniscus act to stretch and immobilize the molecules on a solid surface (6). To manipulate the size, geometry, and alignment of nanowires, much effort has been devoted to controlling the evaporation of the solutions by adjusting experimental parameters, such as concentration and temperature, or applying external forces that move the droplets in a desired direction (7)(8)(9). However, these approaches require careful handling only to generate nanowires that are randomly positioned and oriented.…”

Identifying microbial species by single-molecule DNA optical mapping and resampling statistics

“…Purified DNA was dissolved in 50 mM MES (pH 5.6), and deposited in stretched conformation by mechanically dragging a 2 µl droplet over the surface of a Zeonex-coated coverslip at a speed of 4.4 mm/min using a disposable pipet tip, as described earlier. (28) Stretched samples were stored dry and were vacuum dried overnight prior to imaging.…”

“…This technique causes the DNA fragments to be overstretched by a factor of 1.7 to 1.75. (28) We imaged the labeled DNA with a wide-field microscope. Finally, we extracted the fluorescence intensity signal along the linearized DNA for each individual DNA fragment ( Figure 1C).…”

“…This overstretching of around 75% corresponds to the maximal length of double-stranded DNA before strand-breakage. (28,33) Consequently, overstretching reduces the fluorescent dye overlap probability to 81%. Super-resolved microscopy techniques can further narrow the PSF, thus reducing the overlap probability to 54%.…”

Introductory Chapter: DNA as Nanowires

“…Yet the widespread use is still hindered due to the limited control over the size, geometry, and alignment of the nanowires. Hence to manipulate the size, geometry, and alignment of nanowires, efforts has been needed to control the evaporation of the solutions by adjusting the experimental parameters, such as: concentration and temperature, or by applying external forces that move the droplets in the desired direction [35][36][37].…”