DNA fragments, separation

Southern, E. M., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98 503-517. The classic paper on die idendficadon of specific DNA sequences through hybridizadon widi unique probes. 

Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. JMo/filo/ 1975 98 503-517. 

FIGURE 1 Footp rint analysis of the RNA polymerase-binding site on a DNA fragment. Separate experiments are carried out in the presence (+) and absence (—) of the polymerase. 

Various sieving matrices for DNA fragment separation in a glass chip were evaluated. It was found the performance of HEC was comparable to PDMA, but was superior to polyacrylamide and PEO [611]. Hydroxylpropylcellulose (HPC), instead of HEC or HPMC, was used as a sieving matrix to achieve better DNA separations. In addition, the lower viscosity of HPC allows for more concentrated solution to be used without filling problems. CGE separation of DNA for analyzing mutations associated with Duchenne muscular dystrophy (DMD) was demonstrated [612],... 

FIGURE 9.19 Effect of injection times on steady injection states of different-sized DNA fragments separated on the chip shown in Figure 9.18. The arrows point to the last peaks that reached steady injection states. The separations were performed at ambient temperature by using 4% LPA and an electric field strength of ca. 0.180 V/cm for injection and separation. The data shown were obtained from a channel with an effective separation distance of 7.46 cm [265], Reprinted with permission from the National Academy of Sciences. 

Woolley, A.T., Mathies, R.A., Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips. Proc. Natl. Acad. Sci. USA 1994,91, 11348-11352. 

Specific reports on applications of this technique for forensic purposes are still scarce. The apparent reason for this slow penetration of CE into forensic genetics is twofold. First, the analysis of genetic markers by CE is not a simple technology, and it must be performed in a specialized laboratory. Second, the field of DNA fragment separation by CE is still being developed methodologically and is not an established technique as compared, for example, to slab gel electrophoresis. Nevertheless, this technique should become widespread in forensic biology in the relatively near future. 

Southern, E.M. (1975) Detection of Specific Sequences among DNA Fragments Separated by Gel Electrophoresis, J. Mol. Biol. 98, 503-517. 

DNA fragments separated by polymer networks are detected by ultraviolet (UV) detector or laser-induced fluorescence (LIF) detection. UV detection of DNA fragments is based on the UV absorption of the DNA bases that is, the wavelength and the molar absorption coefficient for the UV absorption maxima of DNA bases are 260 nm for A, 254 nm for G, 267 nm for T, and 271 nm for C, respectively. 

For optimal DNA fragment separation, voltages higher than 125 volts should be avoided. Higher voltages can overheat and melt the gels. 

Mcgregor, D. A. and Yeung, E. S., Detection of DNA fragments separated by capillary electrophoresis based on their native fluorescence inside a sheath flow, J. Chromatogr. A, 680,491,1994. 

Ueno, K. and Yeung, E.S., Simultaneous monitoring of DNA fragments separated by electrophoresis in a multiplexed array of 100 capillaries, AnaZ. Chem., 66, 1424, 1994. 

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