TY - JOUR
AU - Ariano,, Cristiano
AB - To the Editor: Agarose slab gel electrophoresis continues to provide a low-cost alternative and a valuable complement to more sophisticated techniques for estimating the size of DNA molecules after restriction enzyme digestion, for evaluating PCR products, and for separating restricted genomic DNA or RNA before Southern or northern blot analysis (1)(2). With traditional apparatus, the electrophoresis run must be interrupted before the agarose gel can be moved onto a UV transilluminator (wavelength approximately 300–360 nm), which is connected to a conventional or digital camera and placed in a dark environment for best results. This procedure is repeated until a satisfactory separation is achieved. We have devised a simple, easily reproducible device (Fig. 1 ) that contains the following main components: (a) an electrophoresis chamber; (b) 2 UV light–emitting lamps (302 nm), which are placed below the plastic plate holding the gel; and (c) a commercial Web camera connected to a personal computer. The device can be opened to pour or remove a gel and to fill the sample wells. It is configured for connection to an external power supply and is equipped with a safety switch that shuts off the power when the device is opened. The apparatus was built with commercially available materials and may be easily reproduced at a moderate cost. Technical drawings, a list of materials used, and relative costs are available free of charge from the corresponding author. Figure 1. Open in new tabDownload slide Different views of the apparatus for nucleic acid electrophoresis. A list of materials used (with relative costs) and technical drawings are available free of charge from the corresponding author upon request. PC, personal computer. Figure 1. Open in new tabDownload slide Different views of the apparatus for nucleic acid electrophoresis. A list of materials used (with relative costs) and technical drawings are available free of charge from the corresponding author upon request. PC, personal computer. The assembly of these components into a single unit makes our device different from the traditional apparatus; however, its key feature is the part of the electrophoresis chamber holding the gel tray, which is made of UV-transparent Plexiglas (4 mm; Rohm and Haas). This simple modification, combined with the positioning of the UV lamps under the electrophoresis chamber and the availability of a digital camera inside the apparatus, makes it possible to visualize and record separations of nucleic acids without interrupting the run and having to move the gel from the chamber. Our apparatus has 3 substantial advantages compared with traditional devices. First, the visualization and recording of results is shortened and simplified. Digitalizing of the electrophoretic separation is accomplished by simply turning on the video camera and capturing the image. This procedure takes only a few seconds. Second, the separation of DNA molecules is easily optimized. In fact, DNA can be visualized at any moment of the electrophoresis run without interrupting it, allowing selection of the best electrophoretic separation among those recorded. Third, the chemical risks to the operators are reduced. By making unnecessary any movement of the gel in and out of the chamber, use of our apparatus minimizes laboratory contamination and operator contact with the dangerous chemical agents added to the gel (e.g., ethidium bromide), loaded with the samples under analysis (e.g., SYBR® Green I) to make DNA visible under a UV source, or used to denature RNA (formaldehyde). Furthermore, the described apparatus may provide a basis for further configurational developments. In this regard, the possible implementation of recently developed DNA-detection systems based on UV light– or ultrabright green light–emitting diodes could provide further advantages in terms of sensitivity, throughput, and resolving power [e.g., (3)]. A commercial alternative to our apparatus is the FlashGel® system from Lonza Rockland (www.lonza.com). Compared with the FlashGel system, our apparatus has some substantial advantages; in particular, it is possible to carry out prolonged runs for increased separation of DNA fragments through of the use of larger gels [140 mm (length) × 150 mm (width) × 20 mm (height) vs 70 mm (length) × 84 mm (width) × 20 mm (height)] and allowing a free choice of agarose type and concentration for better adaptation of a given protocol to the range of molecular weights of the molecules to be separated; however, the electrophoresis time required for satisfactory separation with our device is at least 4-fold longer than for the commercial device. This disadvantage is partially offset by the fact that our gels can accommodate 80 samples per run (compared with 34 for the FlashGel device). Furthermore, our method is markedly less expensive than the FlashGel device with respect to both the apparatus (613 vs 991 euros) and the agarose gels (0.08 vs 0.37 euros; cost per sample for electrophoresis on a 22-g/L NuSieve® agarose gel from Lonza Rockland). Our apparatus is currently being used for various applications, including checking the size and quality of DNAs extracted from biological materials (e.g., blood and saliva), PCR products of mitochondrial DNA before sequencing, and RFLP typing of polymorphisms within the IRS11 (insulin receptor substrate 1) and MCM6 (minichromosome maintenance complex component 6) genes (4)(5). This newly designed apparatus can be easily reproduced and used in laboratories where analytical and preparative agarose slab gel electrophoresis of nucleic acids is carried out. It has substantial advantages in terms of saving time, optimizing separations, and reducing chemical risks to the operators. Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures of Potential Conflicts of Interest:Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: Ministero dell’Istruzione, dell’Università e della Ricerca, PRIN project protocol no. 2007TYXE3X; Istituto Italiano di Antropologia; and University of Rome “La Sapienza.” Expert Testimony: None declared. Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript. 1 " Human genes: IRS1, insulin receptor substrate 1; MCM6, minichromosome maintenance complex component 6. References 1 Voytas D. Agarose gel electrophoresis. Curr Protoc Immunol 2001 ; 10 : 10.4.1 -8. 2 Brown T, Mackey K, Du T. Analysis of RNA by northern and slot blot hybridization. Curr Protoc Mol Biol 2004 ; 4 : 4.9.1 -19. 3 Liu MS, Amirkhanian VD. DNA fragment analysis by an affordable multiple-channel capillary electrophoresis system. Electrophoresis 2003 ; 24 : 93 -95. Crossref Search ADS PubMed 4 Castrì L, Garagnani P, Useli A, Pettener D, Luiselli D. Kenyan crossroads: migration and gene flow in six ethnic groups from Eastern Africa. J Anthropol Sci 2008 ; 86 : 189 -192. PubMed 5 Coelho M, Luiselli D, Bertorelle G, Lopes AI, Seixas S, Destro-Bisol G, Rocha J. Microsatellite variation and evolution of human lactase persistence. Hum Genet 2005 ; 117 : 329 -339. Crossref Search ADS PubMed © 2009 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - A Simple Homemade Apparatus for Real-Time Visualization of Nucleic Acid Electrophoresis That Saves Time, Optimizes Separation, and Reduces Chemical Risk
JO - Clinical Chemistry
DO - 10.1373/clinchem.2009.125609
DA - 2009-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/a-simple-homemade-apparatus-for-real-time-visualization-of-nucleic-lakVor7O1W
SP - 1436
VL - 55
IS - 7
DP - DeepDyve
ER -