MADGE: Microplate array diagonal gel electrophoresis

JMB 2009; 28 (4) DOI: 10.2478/v10011-009-0030-y UDK 577.1 : 61 ISSN 1452-8258 JMB 28: 235–240, 2009 Review article Pregledni ~lanak MADGE – MICROPLATE ARRAY DIAGONAL GEL ELECTROPHORESIS ELEKTROFOREZA U FORMATU MIKROTITARSKE PLO^E Sanja Stankovi}1, Dragan Alavanti}2, Nada Majki}-Singh1 1Institute 2Laboratory of Medical Biochemistry, Faculty of Pharmacy and Clinical Center of Serbia, Belgrade, Serbia for Radiobiology and Molecular Genetics, VIN^A Institute of Nuclear Sciences, Belgrade, Serbia Summary: Microplate array diagonal gel electrophoresis (MADGE) was invented for molecular genetic epidemiological studies. MADGE is a highly flexible, cost effective, microplate compatible solution to high throughput electrophoresis needs. It enables several thousands to million gel lines per day for direct assay of single-base variation in different capacity laboratories. Variants of the standard 96-well MADGE include: 96-well stretchMADGE, 192-well MADGE, 384-well MADGE, and 768well MADGE. Melt-MADGE combines the temporal thermal ramp apparatus to achieve similar throughput for de novo mutation scanning. Basic MADGE principles and procedures, preparation of MADGE gels, electrophoresis, visualization and analysis of these gels, as well as modifications of the basic 96-well MADGE will be discussed in detail. For the first time in our country, this revolutionary polyacrylamid electrophoresis was done in 1998. We shortly review our studies which used MADGE for high throughput genotyping of the apolipoprotein E. MADGE and melt-MADGE will have an important role in the future genetic research of complex diseases and especially in pharmacogenomics. Keywords: electrophoresis, mutation detection genetic Kratak sadr`aj: »Microplate array diagonal gel electrophoresis« (MADGE) primenjuje se u molekularno genetskim epidemiolo{kim studijama. MADGE je fleksibilna, jeftina metoda, kompatibilna sa formatom standardnih mikrotitarskih plo~a, kojom se mo`e vr{iti elektroforeza nekoliko hiljada-miliona uzoraka dnevno kako u malim tako i u velikim laboratorijama. Postoji nekoliko modifikacija standardne MADGE metode sa 96 bunara i to su »stretch« MADGE, kao i MADGE metode u kojima se koristi ve}i broj bunara (192, 384 i 768). »Melt«-MADGE koristi se za utvr|ivanje novih mutacija. U ovom radu bi}e detaljno prikazani osnovni principi MADGE, priprema gela, elektroforeza, vizuelizacija i analiza tih gelova, kao i modifikacije standardne MADGE metode sa 96 bunara. Ovaj revolucionarni pristup poliakrilamidnoj elektroforezi prvi put je u na{oj zemlji primenjen 1998. godine. U radu }e ukratko biti prikazani rezultati studija u kojima je za genotipizaciju apolipoproteina E kori{}en MADGE. Treba o~ekivati da }e MADGE i »melt«-MADGE imati zna~ajnu ulogu u genetici poligenskih bolesti, posebno u farmakogenomici. Klju~ne re~i: elektroforeza, genetska epidemiologija, detekcija mutacija epidemiology, Introduction Strong evidence from epidemiological and animal studies has implicated genetic influences in the pathogenesis of multifactorial diseases: hypertension, diabetes, ischemic heart disease and cerebrovascular disease. Our knowledge of the three billion base pairs in the human genome could be used to alert patients that they are at risk for certain Address for correspondence: Sanja Stankovi} Institute of Medical Biochemistry, Faculty of Pharmacy & Clinical Center of Serbia, Belgrade, Serbia Tel/Fax: +381 11 3615631 e-mail: sanjastªeunet.rs disease, precisely diagnose disease, and ensure the most effective treatment tailored to patient’s genotype is used and develop new treatments at the molecular level. A major challenge for large-scale studies is to compare hundreds of thousands of polymorphisms among numerous individuals. They also depend on user-friendly technologies that can detect polymorphisms rapidly, accurately, cost effectively in both small and large laboratories. For molecular-genetic epidemiological research, it is important to think about one important step – electrophoresis. It enables ready analysis of size, shape, and charge of molecules. Electrophoresis is also well known and feasible to set up in any laboratory, requiring no expensive hardware. However, gel preparation, long tracks (requiring more cumbersome 236 Stankovi} et al.: MADGE – Microplate array diagonal gel electrophoresis equipment and longer run times), incompatibility with standard microplates, vertical-format polyacrylamide gels (restricting the number of analyses per gel since only one row of samples can be loaded), make electrophoresis an unattractive, but frequently used method in laboratory studies of genetic diversity within populations. Electrophoresis of DNA is traditionally performed either in an improved-quality agarose capable of higher resolution or a polyacrylamide gel matrix. Polyacrylamide still offers the highest resolution for small fragments such as those from PCR and postPCR digests. Agarose gels can easily be prepared in an open-faced format (uncovered, with one or more well-forming combs inserted in the molten substrate) to gain the convenience of horizontal electrophoresis. Acrylamide does not polymerize in the presence of air and the usual configuration is vertical between two glass plates. Microplate array diagonal gel electrophoresis Microplate array diagonal gel electrophoresis (MADGE) was invented by Day in 1994 (1) to enable compatibility between microplate-based liquid phase reactions and polyacrylamide or agarose-gel electrophoretic analysis. This format can be used in optimisation of PCR using gradient thermocyclers. The two-dimensional nature of the electrophoresis system allows the optimal combinations of MgCl2 and temperature to be visualized and compared. MADGE gels are particularly useful in genotyping reactions using restriction fragment linked polymorphism (RFLP) or with amplification-refractory mutation system (ARMS) reaction. The MADGE gel system is based on a standard 96-well microplate array format with a 9 mm well to well distance and 2 mm cubic wells. The array is set on a diagonal of 71.6° giving a final track length of 26.5 mm (2). MADGE gels can be made using two plain glass plates and plastic ‘former’ with 96 2-mm cubic »teeth.« acrylamide content is: for 5% 80-500bp, 7.5% 70– 400bp, 10% 50–300bp. c) pouring the gel mix. It could be done in two different ways. The first is to pour the gel mix onto MADGE gel former, and then place the glass plate, silanized side down, onto former. The second is to place the glass plate over the former, leaving a small gap next to one of the broad borders. Hold the plates together with one hand and tip the plates to a slight incline so that the gap is at the top end. Carefully pour the gel mix into the gap so that it runs down in between the two plates and fills the mould up from the bottom. A 100–250 g weight can be placed centrally on the glass to ensure uniform gel thickness. Once the MADGE gel has set (approximately 15 minutes), ta (...truncated)