History and principles of conductive media for standard DNA electrophoresis.
DNA electrophoresis has been a dominant technique in molecular biology for 30 years. The foundation for this common technique is based on a few simple electrochemical principles. Electrophoretic DNA separation borrowed from existing protein and RNA techniques developed in the 1950s and 1960s. For 30 years, common DNA electrophoretic conductive media remained largely unchanged, with Tris as the primary cation. DNA electrophoresis relies simply upon the negative charge of the phosphate backbone and the ability to distribute a voltage gradient in a sieving matrix. Nevertheless, the conductive properties in DNA electrophoresis are complicated by choices involving voltage, electric current, conductivity, temperature, and the concentration and identity of the ionic species present. Differences among the extant chemical recipes for common conductive media affect central properties. Tris-based buffers, even in optimal form, create a runaway positive feedback loop between heat generation and retention, temperature, conductivity, and current. This is undesirable, leading to limitations on the permissible electric field and to impaired resolution. Recently, we developed low-molarity conductive media to mitigate this positive feedback loop. Such media allow for application of a higher electric field. Applications of DNA electrophoresis can now be reengineered for lower ionic strength, higher field strengths, and lower requirements for heat dissipation.
Brody
Jonathan R
JR
Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at The Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
Kern
Scott E
SE
D002021:Buffers; D004247:DNA / Q000737:chemistry*; D004553:Electric Conductivity; D004586:Electrophoresis / Q000266:history* / Q000379:methods*; D049673:History, 20th Century; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D014325:Tromethamine / Q000737:chemistry*
D002021:Buffers; D014325:Tromethamine; D004247:DNA