HINTS AND TIPS An Evaluation of New Gel Matrices for the Separation of PCR-Amplified DNA Fragments Ramanjini Gowda, Guohao He, Natalie Knight, and Channapatna S. Prakash* Abstract It has been shown that the novel gel matrix, PCR Purity Plus which consists of a vinyl-polymer of poly- acrylamide, provides a superior and rapid means of separating DNA fragments. As this product has been discontinued, the two new commercial versions of this matrix (MDE| and GeneAmp | with PCR Purity Plus were compared. Optimal conditions for resolving DNA fingerprint profiles for both matrices were defined. Both MDE and GeneAmp gels provided a clear separation of DNA fragments. However, the profiles obtained on GeneAmp gel were closest to that of PCR Purity Plus. These results should be useful to DNA fingerprint- ing studies where it is critical to obtain a clear resolution of complex DNA profiles. Index Entries: DNA fingerprinting; polymerase chain reaction; gel matrix; electrophoresis; random amplified polymorphic DNA. Polymerase chain reaction (PCR) has become an indispensable tool in genome research. In stud- ies where PCR is employed to develop DNA fin- gerprint profiles and identify mutants as a result of base-pair differences, it is critical to have a gel system that separates the amplified DNA frag- ments in a reliable manner. We have earlier reported that vinyl-polymer of polyacrylamide provides an improved matrix that clearly resolves PCR-amplified DNA fragments--especiallybetween those with similar sizes and with variable intensi- ties-when compared to either conventional poly- acrylamide or agarose gels (1). This gel matrix was better also because it was easier to handle and tolerates higher voltages and higher temperatures thus allowing faster electrophoresis running time. Recently, however, the commercial supplier of the vinyl-polymer of polyacrylamide (PCR Purity Plus@; AT Biochem, Malvern, PA) has stopped marketing this product. Nevertheless, two more similar gel matrices have been introduced into the market (MDE | by FMC BioProducts, Rockland, ME; and GeneAmp| Detection Gel by Perkin- Elmer, Norwalk, CT). This study follows our earlier report and compares the two newer gel matrices with PCR Purity Plus and identifies the optimal param- eters for resolving DNA fingerprint profiles. This information may be critical to ongoing studies employing PCR Purity Plus where a change of gel matrices is warranted midway through a screening study, and to ensure that such a move would not adversely impact ongoing genome studies. Total genomic DNA from cowpea (Vigna ungui- culata [L.] Walp.) was extracted using a urea-based approach of Chen and Dellaporta (2). Reaction mixes and amplification conditions were followed as described earlier (1). Reaction product aliquots from the same PCR reaction were electrophoresed on PCR Purity Plus, GeneAmp, or MDE gels using a vertical electrophoresis system at 25 V/cm (8 x 10 cm; Mini-Protean II cell; Bio-Rad, Richmond, CA). Gel preparation and electrophoresis conditions were according to manufacturer's recommendations, but it was necessary to modify the proportion of reac- tion components to ensure superior resolution of DNA strands and tailor it to a small gel. DNA was *Author to whom all correspondence should be addressed, Plant Molecular Genetics Lab, Tuskegee University, School of Agriculture and Home Economics, Tuskegee, AL 36088, E-mail: prakash@acd.tusk.edu. Molecular Biotechnology @1996HumanaPress Inc. All rights of any nature whatsoever reserved. 1073-608511996/5:1163-65155.75 MOtECULRR BIOTEHNOtOGV 63 Volume 5, 1996