Single-molecule studies of the interactions of DNA and proteins are important in a variety of biological or biotechnology processes ranging from the protein's search for its DNA target site, DNA replication, transcription, or repair, and genome sequencing. A critical requirement for single-molecule studies is the stretching and immobilization of otherwise randomly coiled DNA molecules. Several methods for doing so have been developed over the last two decades, including the use of forces derived from light, magnetic and electric fields, and hydrodynamic flow. Here we review the immobilization and stretching mechanisms for several of these techniques along with examples of single-molecule DNA-protein interaction assays that can be performed with each of them.

We demonstrate a novel technique we call "protein-assisted DNA immobilization" (PADI), to immobilize and stretch, but not overstretch, DNA molecules inside a micro/nanochannel with limited surface interactions while maintaining continuous hydration at physiological pH. The biological activity of the immobilized DNA molecules is confirmed by digesting the DNA with restriction enzymes in the microchannel. Single-molecule transcription, which has stringent requirements on the immobilized DNA with respect to surface interactions and stretched lengths, is also successfully demonstrated on DNA molecules immobilized by PADI. In addition to arraying DNA molecules for study of DNA-protein interactions, the immobilization method could be used to construct DNA-templated nanoelectronic devices.

Control over the placement of stretched deoxyribonucleic acid (DNA) molecules in a microfluidic system is a critical requirement for molecular nanotechnology. A technique is developed where a large number of DNA molecules can be immobilized specifically at one end to the electrode tip and stretched in a microchannel using high frequency ac fields. λ-DNA molecules are immobilized and stretched using 100kHz ac fields in a 100μm wide and 75μm deep Si microchannel. Using a floating electrode in between two biased electrodes, stretched T2 DNA molecules are immobilized across a 5μm wide electrode gap by electric field and hydrodynamic flow.

Nanoimprint lithography is used to fabricate 200nm–10μm wide SU-8 channels reversal imprinted onto Si substrates. The immobilization and stretching of double stranded λ-DNA molecules within the micro- and nanochannels are demonstrated and controlled by varying the hydrophobicity of SU-8 using oxygen plasma exposure. Site-directed immobilization of DNA is achieved by the integration of 10μm wide SU-8 patterns with 6μm gaps into 100μm wide and 1μm deep Si channels.

Low temperature Si to glass bonding using polymethylmethacrylate (PMMA) as an adhesive layer is developed to integrate electrodes with Si channels. The integrated microsystem contains channels dry etched in Si with widths ranging from 3to100μm and depths ranging from 100nmto30μm. The channels are bonded to a 100μm thick glass consisting of 600nm thick patterned PMMA and 20∕50nm thick Cr∕Au electrodes, with PMMA as an adhesive layer. The typical bond strength is 3MPa, obtained by bonding at 110°C with 600nm thick PMMA. Fluidic flow studies are carried out in channels that are 50 and 100μm wide with a depth of 100nm. De-ionized water flows through the sealed Si channels due to capillary pressure with an initial velocity of 0.65mm∕s for 50μm wide and 100nm deep channels. Electric fields are used to induce DNA motion with velocities from 2.4to14.5μm∕s in 100μm wide and 20μm deep channels. The forces generated by the fields and the fluid flow are also used to stretch the tethered DNA molecules up to 15μm long in the microchannels.