Capillary electrophoresis of DNA fragments in 9 to 20% uncrosslinked polyacrylamide gels: unique separating capacity hypothetically related to maintenance of random-coil DNA conformation independently of gel concentration

Abstract

DNA fragments (0.1 to 2 kb) were separated by capillary electrophoresis (CE) in 9 to 20% uncrosslinked polyacrylamide gels with a resolving power ranging from 3 to 0.1 million theoretical plates/meter across that DNA size range. The unique feature of electrophoresis in 18 to 20% uncrosslinked polyacryalmide is that it provides a method capable of resolving charge isomeric species of DNA fragments (0.4 to 2 kb), confirming a previous report by Heiger et al. [Heiger DN, Cohen AS, Karger BL. J Chromatogr 516 (1990) 33–48]. A similarly unique resolving capacity of uncrosslinked polyacrylamide gels for DNA previously reported is that for heteroduplex DNA [Pulyaeva H, Zakharov SF, Garner MM, Chrambach A. Electrophoresis 15 (1994) 1095–1100] matched by crosslinked gels only in the presence of denaturants [Peeters AV, Kotze MJ. PCR Methods Appl 4 (1994) 188–190; Ganguly A, Rock MJ, Prockop DJ. Proc Natl Acad Sci USA 90 (1993) 10 325–10 329]. A clue as to the cause of that unique resolving capacity of uncrosslinked polyacrylamide is provided by the finding in the present study of a single, gel concentration independent K R [retardation coefficient, d(log mobility)/d(gel concentration)] 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. Electrophoresis 14 (1993) 185–190], it has been interpreted as being due to the transition from a random-coiled to a stretched DNA conformation upon passage through gels of increasing concentration. Since in uncrosslinked gels the decrease of K R does not occur, it is correspondingly assumed that the random-coil conformation of DNA is maintained in those gels in the investigated concentration range up to 20%. The maintenance of random-coil conformation would tend to enhance the resolving capacity of uncrosslinked polyacrylamide because size separation between DNA species is based on K R differences that are maximal in that conformation [Tietz D, Chrambach A. Electrophoresis 14 (1993) 185–190]. The effect of denaturants in allowing for resolution of heteroduplex DNA in crosslinked gels [Peeters AV, Kotze MJ. PCR Methods Appl 4 (1994) 188–190; Ganguly A, Rock MJ, Prockop DJ, Proc Natl Acad Sci USA 90 (1993) 10 325–10 329] supports that hypothesis of the enhanced resolving power of electrophoresis in gels that maintain random-coiled DNA within the gel concentration range used.