344 M. F. zyxwvutsrqpon Arshad zyxwvutsrqponm etal. Electrophoresis zyx 1993, zyx 14, 344-348 Malik F. Arshad' Frederick J. D u n d Raul Vegd Jonathan W. Valvano' Philip Sewer' 'Department of Electrical and Computer Engineering, The University of Texas, Austin, TX 'Instrumentation Services 3Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX Progress in developing improved programs for pulsed field agarose gel electrophoresis of DNA Details are described here for using a rotating gel to perform pulsed field agarose gel electrophoresis (PFGE) with programmable control of the following varia- bles: magnitude of the electrical field, polarity of the electrical field, temperature of the gel and position ofthe rotating disk upon which the agarose gel rests. By use of this procedure for programmable control, modes of PFGE have been explored that have the following characteristics: (i) resolution by DNAlength is completely lost for DNA shorter than a critical length that increases as the pulse times in- crease, and (ii) resolution by DNA length is enhanced for longer DNAs that are shorter than a second critical length. This window of resolution can be moved to the position of the 2-6 Mb chromosomes of zyxw Schizosaccharomyces pombe. 1 Introduction Gel electrophoretic fractionation of DNA is caused by both steric and hydrodynamic interactions zyxwvuts of DNA with the ma- trix of the gel (these interactions are collectively called siev- ing). During pulsed-field gel electrophoresis (PFGE), varia- tion of either the direction or the magnitude of the electri- cal field is used to improve sieving-based fractionations of linear DNA, circular DNA and DNA-protein complexes (re- viewed in [l-71). Pulsing of field can be achieved either by rotating the gel (or electrodes) or by changing the electrical potential on electrodes that surround the gel (reviewed in: [l, 2,4]). The various previously used patterns (modes) of pulsing appear to fall into three categories: (i) the original mode, in which electrophoresis in one direction is periodi- cally alternated with equally long electrophoresis in a sec- ond direction (the angle between the two directions zyxwvu (Y) is between n/2 and zyxwvuts n radians); (ii) the field inversion mode, in which the direction of the electrical field is periodically inverted (i.e., zyxwvutsrqp Y = n radians); and (iii) the rotation-based mode, in which rotation of the field is either conducted at variable speed or periodically interrupted by a period of sta- tionary field (reviewed in [5]). Presumably, other functional modes of pulsing exist, but have not been explored. Addi- tional modes potentially will fractionate DNA longer than the 6-10 Mb DNAs that are the longest DNAs yet fraction- ated by length [8-111. In addition, these modes may im- prove the resolution and flexibility of all DNA fractiona- tions performed by PFGE. Thus, we have constructed a PFGE apparatus that meets the following specifications: (i) complete programmable control over both the direction and the magnitude of the electrical field, and (ii) improved, programmable control over temperature, the parameter whose fluctuations cause the greatest difficulty in maintaining windows of compara- tively high resolution for a restricted range of DNA length. For practical reasons, we have added the additional specifi- cations: (iii) comparatively small footprint, low cost and low amount of heat released into the laboratory, and (iv) access to previously developed programs by use of a menu- driven option. The present communication describes both Correspondence: Dr. P. Sewer, Department of Biochemistry,The Univer- sity of Texas Health Science Center, San Antonio, TX 78284-7760, USA Abbreviation: PFGE, pulsed field gel electrophoresis the software and improvements in hardware for this device. Separation is described for 3-6 Mb DNA subjected to PFGE by use of a mode not previously used. 2 The hardware In a previous report [12], the following components of a sys- tem for PFGE was described: (i) a horizontal, submerged- gel electrophoresis apparatus that performed PFGE by computer-programmed rotation of the gel, (ii) two ther- moelectric Peltier cells that controlled the temperature of electrophoresis. The Peltier cells were attached to the elec- trophoresis apparatus. The electrophoresis apparatus had the shape of a conventional apparatus for submerged hori- zontal gel electrophoresis. Its footprint was only 20-30% greater than the footprint of the electrophoretic bed that contained the gel. This apparatus can be made with any di- mensions (13.3 cm wide,22.0 cm long for those that the au- thors have constructed). For high-volume use, several appa- rati can be placed side-by-side on a counter. Since the previous report, attempts have been made to re- duce the footprint for the control circuitry. All of the cir- cuitry described below has been contained in a rectangular box (43 X 33 X 10 cm) that is vented at the side. Thus, these control boxes can be stacked vertically. The major problem in designing the control box was isolating the control elec- tronics from the varying fields created by switching of the comparatively high current (16 A maximum) of the Peltier cells. The inside of the control box is shown schematically in Fig. 1. The high current component of circuitry for the Peltier cells (A in Fig. 1 is a switching power supply for the Peltier cells) was electromagnetically shielded from the re- maining circuitry by a 1/16 inch aluminum shielding wall (B in Fig. 1). The Peltier cells were controlled by use of an optically coupled solid state relay (C in Fig. 1). This relay electrically isolated the high Peltier cell current from the re- maining circuitry. Figure 1 also shows the following: (i) an Intel 8052-BASIC microprocessor that controls all other components according to the programs described in Sec- tion 3 (D in Fig. l), (ii) an optically isolated circuit to con- trol polarity of the electrical field (E in Fig. l), (iii) a control board for the stepping motor (Fin Fig. 1); (iv) a timing and temperature control board that has two real time clock/ counter integrated circuits (G in Fig. l), (v) a linear power supply for both D and G (H in Fig. l), (vi) vented fans (I in Fig. 1), (vii) vents without fans (J in Fig. l), (viii) an over- @ VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1993 0173-0835/93/0404-0344 $5.00+.25/0