Agarose analysis

There are several forms of electrophoresis. In slab gel electrophoresis the conducting buffer is retained within a porous gel of agarose or polyacrylamide. Slabs are formed by pouring the gel between two glass plates separated by spacers. Typical thicknesses are 0.25-1 mm. Gel electrophoresis is an important technique in biochemistry, in which it is frequently used for DNA sequencing. Although it is a powerful tool for the qualitative analysis of complex mixtures, it is less useful for quantitative work. 

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

Distinction is also made among electrophoretic techniques in terms of the type of matrix employed for analysis. Matrices include polymer gels such as agarose and polyacrjiamide, paper, capillaries, and flowing buffers. Each matrix is used for different types of mixtures, and each has unique advantages. 

Limits of detection become a problem in capillary electrophoresis because the amounts of analyte that can be loaded into a capillary are extremely small. In a 20 p.m capillary, for example, there is 0.03 P-L/cm capillary length. This is 1/100 to 1/1000 of the volume typically loaded onto polyacrylamide or agarose gels. For trace analysis, a very small number of molecules may actually exist in the capillary after loading. To detect these small amounts of components, some on-line detectors have been developed which use conductivity, laser Doppler effects, or narrowly focused lasers (qv) to detect either absorbance or duorescence (47,48). The conductivity detector claims detection limits down to lO molecules. The laser absorbance detector has been used to measure some of the components in a single human cell (see Trace AND RESIDUE ANALYSIS). 

Agarose gels have been used for more than two decades to separate polysaccharides (17-22). In particular, Sepharose CL 2B is widely used (6-8) to separate native starch, but continuously improved mechanical and chemical stability made all of the Sepharose CL gels perfect systems for the analysis of high molecular and broad distributed polysaccharides (23-28). 

Slater, GW Rousseau, J Noolandi, J Tunnel, C Lalande, M, Quantitative Analysis of the Three Regimes of DNA Electrophoresis in Agarose Gels, Biopolymers 27, 509, 1988. 

Presented below are four increasingly stringent confirmatory techniques for PCR and a brief discussion of considerations, limitations and advantages of each. These four techniques are agarose gel electrophoresis, restriction analysis. Southern blotting and sequencing. 

In the previously described electrophoretic methods, the capillary was filled with electrolytes only. Another mode of operation in capillary electrophoresis involves filling the capillary with gel or viscous polymer solutions. If desired, a column can be packed with particles and equipped with a frit.68 This mode of analysis has been favorably used for the size determination of biologically important polymers, such as DNA, proteins, and polysaccharides. The most frequently used polymers in capillary gel electrophoresis are cross-linked or linear polyacrylamide,69 cellulose derivatives,70-75 agarose,76 78 and polyethylene glycols. 

Schmidt, T., Friehs, K., Schleef, M., Voss, C., and Hasche, E., Quantitative analysis of plasmid forms by agarose and capillary gel electrophoresis, Anal. Biochem., 274, 235, 1999. 

Figure 4.1 The comet assay. A single-cell suspension is embedded in agarose on a slide. Cells are then subject to lysis followed by electrophoresis. If present, damaged DNA migrates out of the nucleoid structure during electrophoresis to producing a characteristic comet shape. Double-strand breaks are revealed under neutral conditions, whereas alkali conditions additionally show single-strand breaks and alkali labile sites. Image analysis of stained DNA is used to quantitate the amount of damaged DNA in the comet tail.

---