Microfluidic delivery of cutting enzymes for fragmentation of surface-adsorbed DNA molecules.

We describe a method for fragmenting DNA molecules surface-adsorbed and immobilized onto a polymethyl
methacrylate (PMMA) coated silicon substrate by ‘molecular combing’ [1]. Microfluidic channels were formed into polydimethylsiloxane (PDMS) stamps using the soft lithography technique. The channel widths varied from 3.5 to 7 microns. The channels were pre-filled with a buffer solution by the channel outgas method [2]. Bovine serum albumin was diffused into the channels to coat the PDMS surfaces (which would otherwise strongly adsorb the DNase I cutting enzyme). DNase I was allowed to diffuse into the channel for 1 hour, resulting in efficient and precise fragmentation of the DNAs over the full channel length of 10mm. Applications to DNA sequencing will be discussed.
[1] J. Monahan, A. Gewirth, R. Nuzzo, Anal. Chem. (2001) 73, 3193
[2] A. Bensimon et al, Science (1994) 277, 2096.