agarose gel electrophoresis

Examples of agarose gel electrophoresis in the following topics:

• Genetic Analysis

  • Pulsed field gel electrophoresis (PFGE) is an adaptation of conventional agarose gel electrophoresis that allows extremely large DNA fragments to be resolved (up to megabase size fragments).

• Multiplex and Real-Time PCR

  • It is confirmed by agarose gel electrophoresis for qualitative results.
  • The results from multiplex PCR can be analyzed using gel electrophoresis or using fluorophores for analysis using during the reaction.

• DNA Mobility Shifts

  • A mobility shift assay is electrophoretic separation of a protein-DNA or protein-RNA mixture on a polyacrylamide or agarose gel for a short period.
  • The speed at which different molecules (and combinations thereof) move through the gel is determined by their size and charge, and to a lesser extent, their shape.
  • Under the correct experimental conditions, the interaction between the DNA and protein is stabilized and the ratio of bound to unbound nucleic acid on the gel reflects the fraction of free and bound probe molecules as the binding reaction enters the gel.
  • This stability is in part due to the low ionic strength of the buffer, but also due to a "caging effect"; the protein, surrounded by the gel matrix, is unable to diffuse away from the probe before they recombine.
  • The pattern shown in lane 3 is the one that would result if all the DNA were bound and no dissociation of complex occurred during electrophoresis.

• Northern Blots

  • Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence.
  • The term 'Northern blot' actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane.
  • RNA samples are then separated by gel electrophoresis.
  • Since the gels are fragile and the probes are unable to enter the matrix, the RNA samples, now separated by size, are transferred to a nylon membrane through a capillary or vacuum blotting system.

• Precipitation Reactions

  • These can be performed in semisolid media such as agar or agarose, or non-gel support media such as cellulose acetate.
  • Precipitation methods include double immunodiffusion (qualitative gel technique that determines the relationship between antigen and antibody), radial immunodiffusion (semi-quantitation of proteins by gel diffusion using antibody incorporated in agar), and electroimmunodiffusion (variation of the double immunodiffusion method reaction that uses an electric current to enhance the mobility of the reactants toward each other).
  • The Mancini method results in precipitin ring formation on a thin agarose layer.

• DNA Protection Analysis

  • The method uses an enzyme, deoxyribonuclease (DNase, for short) to cut the radioactively end-labeled DNA, followed by gel electrophoresis to detect the resulting cleavage pattern.
  • Cleavage by DNase will produce fragments, the smaller of which will move further on the electrophoretic gel.
  • The fragments which are smaller will appear further on the gel than the longer fragments.
  • The gel is then used to expose a special photographic film.
  • This protection will result in a clear area on the gel which is referred to as the "footprint".

• Immunoblot Procedures

  • Proteins are generally separated by size using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis.
  • As the proteins migrate out of the gel, they are captured on a membrane.
  • In clinical diagnostic settings, immunoelectrophoresis is applied, which involves the electrophoresis of serum or urine followed by immunodiffusion.

• DNA Sequencing Based on Sanger Dideoxynucleotides

  • Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis.
  • This is frequently performed using a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C).
  • The DNA bands may then be visualized by autoradiography or UV light and the DNA sequence can be directly read off the X-ray film or gel image.
  • DNA sequencers carry out capillary electrophoresis for size separation, detection and recording of dye fluorescence, and data output as fluorescent peak trace chromatograms.

• Pure Culture

  • For the purpose of gelling the microbial culture, the medium of agarose gel (agar) is used.

• Western Blots

  • The technique uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide.