A Low-Cost, High-Throughput Polyacrylamide Gel Electrophoresis System for Genotyping with Microsatellite DNA Markers

staining. However, these visualization methods require either expensive or hazardous radioactive chemicals and Microsatellite DNA markers are widely used in genetic research. are time-consuming. Electrophoresis with MetaPhor Their use, however, can be costly and throughput is sometimes limited. The objective of this paper is to introduce a simple, low-cost, highagarose gels (Cambrex Corporation, East Rutherford, throughput system that detects amplification products from microsaNJ) has been used to separate alleles of microsatellite tellite markers by nondenaturing polyacrylamide gel electrophoresis. markers, but the resolution is lower than nondenaturing This system is capable of separating DNA fragments that differ by polyacrylamide gels and the cost is currently five times as little as two base pairs. The electrophoresis unit holds two vertical more than that of nondenaturing polyacrylamide gels. 100-sample gels allowing standards and samples from a 96-well plate Capillary electrophoresis also has been used to deterto be analyzed on a single gel. DNA samples are stained during mine length polymorphisms of microsatellite markers electrophoresis by ethidium bromide in the running buffer. In addi(Marino et al., 1995), but this method requires sophistition, one of the gel plates is UV-transparent so that gels can be cated instruments and fluorescently tagged primers, photographed immediately after electrophoresis without disassemwhich are expensive. Here we describe an inexpensive bling the gel-plate sandwich. Electrophoresis runs are generally less than two hours. The cost per gel, excluding PCR cost, is currently and relatively high-throughput system developed for the estimated at about $2.60, or less than $0.03 per data point. This system purpose of genotyping with microsatellite markers. has been used successfully with soybean [Glycine max (L.) Merr.] and wheat (Triticum aestivum L.) microsatellite markers and could MATERIALS AND METHODS be a valuable tool for researchers employing markers in other species. The Electrophoresis Unit The electrophoresis unit was custom-designed and manuM markers, also referred to as simple factured by C.B.S Scientific Co. (Del Mar, CA) according to sequence repeat (SSR) DNA markers, are widely our requirements (Fig. 1). C.B.S Scientific Co. now markets employed in linkage map construction, quantitative trait this “MEGA-GEL High Throughput Vertical Unit” (model C-DASG-400-50). The unit holds two gels vertically and has loci (QTL) mapping, and the analysis of genetic divera rotating base for easy access to both gels. The two gels can sity (Wang et al., 2001; Cregan and Quigley, 1997; Tembe run either simultaneously or independently. The gels are nykh et al., 2000; Roder et al., 1998; Marino et al., 1995). cast and run between glass plates that are 50 cm wide and Microsatellites are direct tandem repeated sequences 22 cm high. The back plate (round-cornered plate) is UV of DNA with a repeat size ranging from one to six transparent so that gel photography can be done without disasbase pairs. Microsatellites have been found to be both sembling the gel-plate sandwich. Each gel has the capacity for abundant and widely distributed throughout the ge100 samples so that all samples from a 96-well plate can be nomes of many higher plants and animals (Burow and loaded in one gel. The sample wells are spaced 4.5 mm apart Blake, 1998). The number of tandem repeats in a microfor loading with a standard 8or 12-channel pipette. satellite is highly variable among individuals in a species and is the basis for the length variability of the marker. The Protocol The length polymorphism of a microsatellite marker The nondenaturing polyacrylamide gel is cast in a glass is commonly detected through polymerase chain reacplate sandwich (Fig. 1) with a 1.5-mm-thick spacer on each tion (PCR) amplification using primers flanking the mivertical side, a gel-wrap gasket (Catalogue No. VGE-15XX, crosatellite, followed by electrophoresis to determine CBS Scientific, Del Mar, CA), and six spring clamps. The gelthe length of the PCR product. There are several separawrap gasket is mounted on the bottom and side edges of the tion methods currently employed to determine the back plate and sandwiched between the two glass plates to form a leak-proof seal. The gasket replaces the need for tape. length of amplification products among which polyacrylThe glass plates are cleaned with the household glass cleaner amide gels are commonly used. The amplification prodWindex (SC Johnson, Racine, WI) and wiped with disposable ucts in polyacrylamide gels are typically visualized with low-lint Kimwipes (Kimberly-Clark, Dallas, TX). The plate radioactive labeling, fluorescent dye labeling, and silver sandwich is assembled similar to other polyacrylamide gels with spacers and clamps. Two medium office binder clips are D. Wang, J. Shi, and R.W. Ward, Dep. of Crop and Soil Sciences, placed at the plate sandwich bottom to provide a tight gasket Michigan State University, East Lansing, MI 48824; S.R. Carlson and seal and provide additional support during pouring. The plate B.W. Diers, Dep. of Crop Sciences, 1101 W. Peabody Dr., University sandwich stands upright using the binder and spring clamps of Illinois at Urbana-Champaign, Urbana, IL 61801; P.B. Cregan, at the bottom for support. Soybean Genomics and Improvement Laboratory, USDA-ARS, Approximately 180 mL of gel solution is needed to cast BARC-West, Beltsville, MD 20705. Received 18 Dec. 2002. *Correeach gel. The final concentration of each gel is 6% (w/v) sponding author ([email protected]). Abbreviations: bp, base pair; ITMI, International Triticeae Initiative; Published in Crop Sci. 43:1828–1832 (2003).  Crop Science Society of America PCR, polymerase chain reaction; QTL, quantitative trait loci; SSR, simple sequence repeat. 677 S. Segoe Rd., Madison, WI 53711 USA