Electrophoresis-II

Polyacrylamide Gels

Instrumentation

Separation

Detection





Separation of DNA/RNA

Electrophoretic techniques are important tools for identiﬁcation and sequencing of DNA and RNA.Electrophoretic techniques can commonly be used for separation of linear fragments from 20 to 20000 bp (base pairs). However with special pulse techniques, DNAs with up to 5x106bp can be separated. Polyacrylamide gels are used for DNA fragments up to 1000bp, while agarose gels are used for larger molecules. In a neutral or slightly basic environment, DNA molecules have a net negative charge distributed along the molecule, and the charge/size ratio will be approximately the same for all DNA molecules/fragments. Hence, without any sieving effect in the gel, all fragments will travel with the same velocity, and no separation is obtained.By using a gel with appropriate pore size giving a sieve effect for the fragments to be separated, separation can be achieved. The shorter DNA fragments will migrate faster than the longer ones in the gel. DNA fragments are usually tagged with a ﬂuorescent compound before the separation for easy detection.





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Polyacrylamide is a well-deﬁned,stable,and rather inert gel with a pore size that can be easily varied. It is mechanically strong, easily handled, and transparent. Due to relatively small pore size, the eddy diffusion is low and hence contributes little to band broadening. However, due to the small pore size, polyacrylamide gels are less suitable for separation of very large molecules such as proteins with molecular mass >300000 (300kDa). Polyacrylamide gels are made by polymerization of acrylamide (toxic) with N,N'-methylenebisacrylamide in the presence of free radicals generated from either ammonium persulfate (oxidative chemical initiator) or riboﬂavin-50-phosphate (photochemical initiator). The reaction is controlled by equimolar concentrations of N,N,N',N'-tetramethylethylene diamine (TEMED) as catalyst.The pore size is characterized by %T (total acrylamide concentration) and %C:%T =(a+b/m) *100%and%C =(b /a+b)*100%;where a is grams of acrylamide, b is grams of N,N'-methylenebisacrylamide, and m is the total gel volume in milliliter. For example, a typical 19+1(acryl + amideþN,N'-methylenebisacrylamide) gel has %T=8% and %C=5%.In polyacrylamide gel separations, a discontinuity in the gel and/or electrolyte composition (pH) can give rise to a concentration of the start band prior to the separation, giving better efﬁciency and hence resolution.Different types of instrumentation are available, depending on the kind of matrix used as carrier for the electrolyte. The separations can be carried out in columns (vertically) or on thin layers (vertically or horizontally). For analytical separations, thin layer separation systems are the most common.Water cooling is used to provide constant temperature,and is especially important for high-voltage electrophoretic separations. Common dimensions of the thin layer are 5–20cm x 5–20cm and the layer thickness is 0.1–2mm for polyacrylamide gels and 5–10mm for agarose gels (for analytical purposes).Sample ApplicationSample application is performed by introducing the samples into the gel before applying voltage for separation. Several samples and standards can be applied to the same gel. The samples and standards are introduced into separate wells, which are located in the gel on a line parallel to the side. The wells in the gel are made when the gel is prepared by placing a form (comb) with a deﬁned (inverse) slot size.Typical size of the wells is 1mm x 5mm, while the depth must be less than the thickness of the gel. The sample volume applied is typically 5–50ml.The separation starts when the electric ﬁeld is applied. Each compound travels as a band with a velocity, which generally depends on its charge/size ratio. Identiﬁcation of analytes can be done by comparing the migration length with that of standards separated in the same gel (applied in a separate well).Detecting the separated compounds can be challenging in gel electrophoretic techniques. Detection of non colored compounds can be performed by addition of reagents to the gel that react with the compounds to form colored derivatives.This is called staining. Excess reagent often needs to be removed by washing or electrophoretically, and this is called destaining. In some cases when the staining cannot be performed directly in the gel, the analytes are transferred to another medium, for example, a nitrocellulose membrane, by contact diffusion or electrophoretically and detected by staining. To document the obtained visualized separation, the gel (or secondary medium) is photographed. DNA fragments and proteins can be tagged with a ﬂuorescentcompoundbeforetheseparation.With UV light irradiating the gel after completed separation,the compounds can be detected by their ﬂuorescence.In this case, photographic documentation is required. Documentation can also be performed with a densitometer, scanning the whole gel and obtaining information of the intensity as a function of location. The densitometer can also be used for quantitative determinations.