CAPILLARY ELECTROPHORESIS OF DNA FRAGMENTS IN 9-PERCENT TO 20-PERCENTUNCROSSLINKED POLYACRYLAMIDE GELS - UNIQUE SEPARATING CAPACITY HYPOTHETICALLY RELATED TO MAINTENANCE OF RANDOM-COIL DNA CONFORMATION INDEPENDENTLY OF GEL CONCENTRATION

DNA fragments (0.1 to 2 kb) were separated by capillary electrophoresi s (CE) in 9 to 20% uncrosslinked polyacrylamide gels with a resolving power ranging from 3 to 0.1 million theoretical plates/meter across th at DNA size range. The unique feature of electrophoresis in 18 to 20% uncrosslinked polyacryalmide is that it provides a method capable of r esolving charge isomeric species of DNA fragments (0.4 to 2 kb), confi rming a previous report by Heiger et al. [Heiger DN, Cohen AS, Karger BL. J Chromatogr 516 (1990) 33-48]. A similarly unique resolving capac ity of uncrosslinked polyacrylamide gels for DNA previously reported i s that for heteroduplex DNA [Pulyaeva H, Zakharov SF, Garner MM, Chram bach A. Electrophoresis 15 (1994) 1095-1100] matched by crosslinked ge ls only in the presence of denaturants [Peeters AV, Kotze MJ. PCR Meth ods Appl 4 (1994) 188-190; Ganguly A, Rock MJ, Prockop DJ. Proc Natl A cad Sci USA 90 (1993) 10 325-10 329], A clue as to the cause of that u nique resolving capacity of uncrosslinked polyacrylamide is provided b y the finding in the present study of a single, gel concentration inde pendent K-R [retardation coefficient, d(log mobility)/d(gel concentrat ion)] for the DNA fragments, which contrasts with the decrease of K-R with gel concentration observed for crosslinked polyacrylamide across a wide concentration range [Orban L, Chrambach A. Electrophoresis 12 ( 1991) 241-246; Tietz D, Chrambach A. Electrophoresis 14 (1993) 185-190 ]. Since the decrease of K-R with gel concentration correlates with a decrease in equivalent molecular radius [Tietz D, Chrambach A. Electro phoresis 14 (1993) 185-190], it has been interpreted as being due to t he transition from a random-coiled to a stretched DNA conformation upo n passage through gels of increasing concentration. Since in uncrossli nked gels the decrease of K-R does not occur, it is correspondingly as sumed that the random-coil conformation of DNA is maintained in those gels in the investigated concentration range up to 20%. The maintenanc e of random-coil conformation would tend to enhance the resolving capa city of uncrosslinked polyacrylamide because size separation between D NA species is based on K-R differences that are maximal in that confor mation [Tietz D, Chrambach A. Electrophoresis 14 (1993) 185-190]. The effect of denaturants in allowing for resolution of heteroduplex DNA i n crosslinked gels [Peeters AV, Kotze MJ. PCR Methods Appl 4 (1994) 18 8-190; Ganguly A, Rock MJ, Prockop DJ, Proc Natl Acad Sci USA 90 (1993 ) 10 325-10 329] supports that hypothesis of the enhanced resolving po wer of electrophoresis in gels that maintain random-coiled DNA within the gel concentration range used. (C) 1997 Elsevier Science B.V.