5. Electrophoresis

Electrophoresis is a technique used in the laboratory that results in the separation of charged molecules. \\"Gel\\", refers to the matrix used to separate the molecules. Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemistry. Gel electrophoresis is the separation of deoxyribonucleic acid, ribonucleic acid, and protein through an electric charge. It is usually performed for analytical purposes, but may be used as a preparative technique to partially purify molecules prior to use of other methods such as mass spectrometry, PCR, cloning, DNA sequencing, or immuno-blotting for further characterization.

Applications of agarose electrophoresis are: Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction mapping of cloned DNA; Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting; And separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer. Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to size fractionate DNA molecules, which then could be eluted from the gel.

The advantages are that the gel is easily poured, does not denature the samples, and is physically firmer than polyacrylamide. The samples can also be recovered. Applications of polyacrilamide electrophoresis are: Electrophoresis, refers to the electromotive force that is used to push or pull the molecules through the gel matrix; by placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, towards the anode if negatively charged or towards the cathode if positively charged (note that gel electrophoresis operates as an electrolytic cell; the anode is positive and the cathode is negative). In the case of DNA, the direction of migration, from negative to positive electrodes, is due to the natural negative charge carried on their sugar-phosphate backbone.

Gel electrophoresis of large DNA is usually done by agarose gel electrophoresis. The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device. The image is recorded with a computer operated camera, and the intensity of the band is measured and compared against standard or markers loaded on the same gel. The measurement and analysis are mostly done with specialized software. Polyacrylamide gel electrophoresis is used for separating DNA fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins. The exact distance between bands is influenced by percentage of agarose, time of electrophoresis, concentration of Ethidium bromide, degree of supercoiling and the size and complexity of the DNA.

Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. The higher the voltage, the faster the DNA migrates. But voltage is limited by the fact that it heats and ultimately causes the gel to melt. High voltages also decrease the resolution (above about 5 to 8 V/cm). Shorter molecules move faster and migrate further than longer ones. Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their radius of gyration, or, for non-cyclic fragments, roughly size. Single-stranded DNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with different speeds (slowest to fastest): nicked or open circular, linearised, or supercoiled plasmid.

Collaborators

  • Smithsonian
  • UNIPA
  • UB
  • IBRC

Fundings:

  • USAID