There are two general types of problem that are readily solved by electrophoretic techniques in the molecular laboratory. The first is the need to accurately assess the size of DNA fragments; the second is the need to identify both characterized and uncharacterized mutations or sequence variants.
5.1. SIZING OF DNA FRAGMENTS BY ELECTROPHORESIS The need to determine the size of DNA fragments is a common occurrence in the molecular laboratory. Examples include verification of the identity of cloned DNA fragments by restriction enzyme digestion, determination of the size of a band detected by Southern transfer, and verification of the size of a PCR product.
The size range of DNA fragments separable by any gel system is a function of the pore size of the matrix [this parameter is related to the viscosity of the matrix shown in Eq. (2) of Table 1]. The pore size is primarily a function of concentration in agarose gels and %T and %C in acrylamide gels. Even though the optimum separation ranges for different gels vary widely, they all have the same general profile. For all gels, there will be a region of optimal separation that is proportional to the log of the size of the DNA fragment. Similarly, there will be a point at which all DNA fragments are smaller than the effective pore size and will be not be retarded by the gel matrix and a point at which all DNA fragments are too large for any of the gel pores. At these two points, mobility is independent of molecular size, and all DNA fragments above or below these limits comigrate. In the laboratory, size determinations are typically made by comparison to a size marker that has been run on the same gel as the unknown sample. A variety of size markers containing DNA fragments of known length are commercially available. A standard curve is prepared by plotting the log of the migration distance of each band in the size marker vs its size in basepairs. The migration distance of the unknown band is plotted and the size of the fragment read off the graph.
After the discovery of trinucleotide repeat expansion as a mechanism for genetic disease, the molecular diagnostics laboratory had a need for high-resolution DNA fragment sizing. In this case, PCR products prepared with primers flanking the triplet repeat region of interest are electrophroresed on a highresolution polyacrylamide gel or analyzed by CE. In the slab gel method, the high resolution is achieved by conducting the electrophoresis on a long (30-40 cm long), thin (0.4-0.6 mm thick) polyacrylamaide gel. In this technique, essentially identical to DNA sequencing methods, the PCR products are typically radiolabeled with 32P or 33P and detected by autora-diography. Figure 4 shows an example of a typical analysis of the Fragile X locus. In the CE method, one of the PCR primers is labeled with a fluorescent tag, and the sizing is done in reference to an internal standard, which consists of DNA fragments of known lengths that are labeled with a different fluorescent molecule. Figure 5 shows an example of a typical analysis of the spinocerebellar ataxia type I locus.
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