Electrophoresis

There two main techniques by which electrophoresis is achieved today: gel and capillary electrophoresis, both of which are based on the same principle of size and charge based fragment separation. This separation is facilitated by the negative charge present on the DNA fragments due to the release of positive hydrogen ions from the phosphate groups that constitute the 'backbone' of the molecule in the presence of ionic buffer solutions such as Tris Borate Ethylenediamine tetra–acetic acid (EDTA) (TBE) or Tris Acetate EDTA (TAE).

Gel electrophoresis

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Gel electrophoresis can be performed in a horizontal or vertical plane, using agarose or polyacrylamide gel as a separation medium. Despite the variation used, the presence of an ionic buffer solution and constant electrical charge across the gel is a ubiquitous necessity in achieving separation of the DNA.

Gels are usually made in the laboratory by pouring the liquid form of either agarose or polyacrylamide into a pre-formed solid mould; a comb is inserted at one end to create wells, into which the DNA of interest will be loaded for separation. The gel is then allowed to solidify before the comb is removed and the gel is transferred into a buffer-containing electrophoresis tank where the DNA can be loaded into the gel wells and separation can take place.

Agarose gels

The choice of gel used is largely dependent upon the size and spacing of the DNA fragments under analysis. Agarose is a polysaccharide which, together with agaropectin, forms the seaweed-derived, gelatinous substance agar. When set, the polysaccharide strands form a matrix structure through which DNA molecules can travel when a charge is present at either end of the gel.

Agarose gel

The pore sizes within this matrix are considered relatively large at approximately 100-300nm depending upon the concentration of agarose, and as such it does not allow for accurate resolution of closely sized DNA fragments. Agarose gels are generally used when larger fragments, in the region of 500-20,000bp, are required to be visualised.

They can also be used to assess the quality of extracted DNA, with degraded template producing a smear when run on an agarose gel as opposed to a tight band of high molecular weight for high quality samples. Agarose gel electrophoresis is used for fragment separation during the DNA fingerprinting method described above.

Polyacrylamide gels

Polyacrylamide gels are made by inducing polymerisation of acrylamide and bisacrylamide monomers in a process initialised by the presence of Ammonium Persulphate and TEMED (N, N, N', N'-tetramethylethylenediamine).

The use of an artificial gel matrix in place of the naturally extracted agarose produces smaller pore sizes in the gel matrix at approximately 10-20nm in a typical gel. This pore size reduction, along with optimised running conditions, can allow for highly accurate resolution of similarly sized DNA fragments and under denaturing conditions achieves resolution of single base-pair size differences.

Polyacrylamide gel

This level of accuracy led to polyacrylamide gels being employed for separation of amplified STR markers during DNA profiling development but has now been largely replaced by more sensitive capillary electrophoresis technologies.

Polyacrylamide gel electrophoresis (PAGE) is conducted in a manner very similar to agarose gel analysis. The un-polymerised solution is poured between two closely spaced glass plates, a gel comb is inserted to create the wells into which the DNA will be loaded and the solution is allowed to polymerise or set over 1-2 hours.

Once set, the gel is moved to the running apparatus where a buffer is placed at the top and bottom of the gel, the DNA of interest is loaded into the wells created during polymerisation, and separation occurs in the same way as for agarose gels when a fixed current is applied across the gel apparatus.

Capillary electrophoresis

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Capillary electrophoresis (CE) is more accurately described as a variation on the more established gel electrophoresis methods rather than a new technique in its own right. The main difference in the two electrophoretic techniques is the use of a capillary containing a polymer solution such as hydroxyethylcellulose in place of the traditional physical gel.

3130 Genetic analyser

Capillaries are made of fused silica and have an internal diameter of only 50-100μm and can be 25-100cm in length. Similarly to gel electrophoresis, size separation is achieved via use of buffer solutions and application of positive and negative charges at either end of the capillary.

Another major difference is in the method of loading the DNA sample of interest in to the capillary. With gel systems the samples must be carefully loaded by an operator into the wells created during casting of the gel. For CE, the DNA sample is loaded into the separation medium by electrokinetic injection whereby a positive charge is applied to draw the negatively charged DNA into the capillary. This method of loading requires less operator time and is very amenable to automated and high-throughput processing.

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