Separation of Large DNA Molecules

Single A.DNA molecules (41 pM) were detected as bursts on-chip using the intercalating dye YOYO-1 [159]. Single molecule detection of A.DNA (310 fM) and other small DNA molecules was also performed on PC or PMMA chips, using TOPRO-5 [986], 

FIGURE 9.23 Optical micrograph of a T-channel device used for DNA separation. The 

FIGURE 9.24 The device consisting of a Brownian ratchet array for DNA separation. The flow angle, 0IMl, is 10.8° with respect to the vertical array axis. //, L, and L were 6, 8, and 10 pm, respectively. The obstacles were 5.6 pm long, 1.4 pm wide, and 3.2 pm tall [686]. Reprinted with permission from the American Chemical Society. 

DNA separation was also achieved in nanostmctured regions that were constructed by magnetic beads (700 nm). These beads were self-assembled with 5.7-pm spacings in the presence of a magnetic field. Then separation of DNA molecules (15, 33.5, and 48.5 kbp) was conducted in such an environment [993,994]. In another report, separations of X and T4 DNA was conducted in a sieving matrix formed by smaller-sized magnetic beads (570 nm) which were self-assembled in a PDMS chip [995]. 

FIGURE 9.30 Fluorescence images of (a) a XI)NA and (b) a T4 DNA migrating in a nanopiUar region at 7 V/cm [351]. Reprinted with permission from the American Chemical Society. 

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Carle, G. Frank, M. Olson, M. Electrophoretic separation of large DNA molecules by periodic inversion of the electric field. Science 1986 232 65-68. 

Agarose and polyacrylamide gels are the most common used matrixes for the separation of nucleic acids. Small size nucleic acids (less than 2 kb) are usually separated on agarose gels. For high resolution and for the separation of large DNA molecules polyacrylamide gel is recommended. Polyacrylamide gels are also used for the separation of proteins. 

Olson, M. (1989). Separation of large DNA molecules by pulsed-field gel electrophoresis. A review of the basic phenomenology. J. Chromatogr. 470, 377-383. 

Chu G., VoUrath D., and Davis R.W. 1986. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234 1582-1585. 

Pulsed field gel electrophoresis A type of gel electrophoresis in which the orientation of the electric field is charged periodically. This technique makes it possible to separate very large DNA molecules, up to the size of whole chromosomes. 

Schwartz and Cantor developed pulsed field gel electrophoresis for the separation of very large DNA molecules. 

Capillary Gel Electrohoresis. In this mode, molecules are separated according to size as they migrate through a polymer matrix. The polymer can be in solution, physically coated on the capillary wall, or chemically bonded to the capillary wall. This mode is primarily used for the separation of large molecules like proteins, peptides, and DNA species. 

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