Gel Electrophoresis
Molecular Diagnostics
(MDx) Review 563
Principle
Matrices
Buffers
Tracking dyes
Stains
Variations
Applications
Nucleic acids migrate to pos pole (anode) in electrical field due to neg charge on phosphate group. Molecules with lowest
MW travel furthest from point of application. Traditional method to detect/analyze nucleic acids following isolation.Agarose, polyacrylamide.Tris-acetate-EDTA (TAE), tris-borate-EDTA (TBE).Bromphenol blue, xylene cyanol.Ethidium bromide (fluorescent DNA-binding dye, mutagen, handle with care), silver, Coomassie blue.Polyacrylamide gel electrophoresis (PAGE): Used when DNA fragments are small. High degree of resolution. Can differentiate
fragments that differ by just 1 base pair.Capillary electrophoresis: Performed in thin capillary tubes using high voltage. Used in DNA sequencing & fragment
analysis.Pulsed-field gel electrophoresis: Alternates direction of current. Used in epidemiology of infectious diseases.Denaturing gel electrophoresis: Uses chemicals to denature secondary structures. Used most often in electrophoresis
of RNA.PCR/RFLP, RT-PCR fragment analysis, Southern & Northern blotting, DNA sequencing.