Separation of Large Circular DNA by Electrophoresis in Agarose Gels

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• 作者：Kenneth D. Cole and Carlos M. Tellez
• 刊名：Biotechnology Progress
• 出版年：2002
• 出版时间：February 2002
• 年：2002
• 卷：18
• 期：1
• 页码：82 - 87
• 全文大小：77K
• 年卷期：v.18,no.1(February 2002)
• ISSN：1520-6033

文摘

The electrophoresis of circular DNA, ranging in size from 4.4 kilobase pairs (kbp) to220 kbp, was studied in agarose gels. Bacterial artificial chromosome (BAC) DNA wasused as a source of large supercoiled and open circular (relaxed) forms. The open circlesabove approximately 50 kbp were trapped at the sample wells of 1% agarose gels duringelectrophoresis at 3 V/cm. Field inversion gel electrophoresis (FIGE) was used to relievethe 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 gelvelocity of the open circles was measured at approximately 20 kbp. Open circles belowapproximately 20 kbp migrated slower than the supercoiled forms, and above 20 kbpthe order was reversed. These results indicate that when the size of the open circlesexceeded the average pore size of a gel and it was forced to span multiple pores, theopen circles gained a mobility advantage. Decreasing the ionic strength of theelectrophoresis buffer significantly decreased the mobility of the smaller circles andslightly increased the mobility of the larger circles.