Pulsed field separation of large supercoiled and open-circular DNAs and its application to bacterial artificial chromosome cloning.
We have studied the separation of large (80-300 kbp) supercoiled (SC) DNA in conventional agarose gel electrophoresis, field inversion gel electrophoresis (FIGE) and pulsed field gel electrophoresis (PFGE). DNA migration was measured under a variety of electrophoretic conditions including different switch times, temperatures, agarose concentrations, and voltage gradients. The migration of SC DNA was found to be inversely proportional to its molecular weight in the three electrophoresis systems tested. In conventional agarose electrophoresis, voltage gradient was found to be the determining parameter in the separation of SC DNA. Unlike large linear DNAs, the migration of SC DNA was found to be independent of switch time in PFGE and FIGE. Broad DNA bands were observed in prolonged FIGE runs. In addition, we have also studied the migration of open-circular (OC) DNA (80 and 100 kbp) in pulsed field gel electrophoresis. Eighty kbp OC DNA can migrate into agarose gels under certain pulsed field conditions whereas 100 kbp OC DNA was trapped at the wells. Based on electrophoretic conditions described in this report, we can determine the size of bacterial artificial chromosome (BAC) clones without restriction enzyme digestion and have enriched the percentage of larger size clones in BAC cloning.
Wang
M
M
Department of Pharmacology, University of North Carolina at Chapel Hill 27599-7365, USA.
Lai
E
E
D000818:Animals; D016466:CHO Cells; D002876:Chromosomes, Bacterial; D002889:Chromosomes, Human, Pair 2; D003001:Cloning, Molecular; D006224:Cricetinae; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004270:DNA, Circular / Q000302:isolation & purification*; D004278:DNA, Superhelical / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D006801:Humans; D013696:Temperature; D013997:Time Factors
D004269:DNA, Bacterial; D004270:DNA, Circular; D004278:DNA, Superhelical