Separation of large circular DNA by electrophoresis in agarose gels.
The electrophoresis of circular DNA, ranging in size from 4.4 kilobase pairs (kbp) to 220 kbp, was studied in agarose gels. Bacterial artificial chromosome (BAC) DNA was used as a source of large supercoiled and open circular (relaxed) forms. The open circles above approximately 50 kbp were trapped at the sample wells of 1% agarose gels during electrophoresis at 3 V/cm. Field inversion gel electrophoresis (FIGE) was used to relieve the trapping of the open circles in the gels. Using FIGE (30 s forward pulse time), open circles with sizes of 115 and 220 kbp required reverse pulse times of 3 and 6 s, respectively, to free the circles from open-ended gel fibers. A minimum in the gel velocity of the open circles was measured at approximately 20 kbp. Open circles below approximately 20 kbp migrated slower than the supercoiled forms, and above 20 kbp the order was reversed. These results indicate that when the size of the open circles exceeded the average pore size of a gel and it was forced to span multiple pores, the open circles gained a mobility advantage. Decreasing the ionic strength of the electrophoresis buffer significantly decreased the mobility of the smaller circles and slightly increased the mobility of the larger circles.
Cole
Kenneth D
KD
Bioprocess Engineering Group, Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA. Kenneth.Cole@nist.gov
Tellez
Carlos M
CM
D002021:Buffers; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; 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; D008970:Molecular Weight; D009994:Osmolar Concentration
D002021:Buffers; D004270:DNA, Circular; D004278:DNA, Superhelical