Electrophoretic Karyotyping

Abstract

For most fungi, cytological investigations to determine chromosome number and sizes, called karyotyping, are precluded because of the small size of fungal chromosomes. Therefore, time-consuming crossing experiments have been performed to characterize the fungal genome and to construct linkage maps. Imperfect species without sexual reproduction were accessible only to studies of mitotic recombination events. For these reasons, the isolation and electrophoretic separation of intact chromosomes would greatly facilitate karyotyping of fungal genomes. Unfortunately, DNA molecules larger than about 50 kilobases (kb) cannot be subjected to standard gel electrophoresis. Due to the negative charge, DNA molecules migrate under the influence of an electric field through the gel which acts as a sieving medium. Using agarose gels, the mobility of DNA molecules larger than about 30 kb rapidly decreases and, as a result, discrete bands are not resolved. Even the use of lower agarose concentrations resulting in a larger mesh size does not allow the separation of these molecules. This size-independent mobility is explained by the biased reptation model (Lumpkin et al. 1985; Slater et al. 1987). In 1984, Schwarz and Cantor introduced a novel electrophoresis system, capable of separating DNA molecules of up to 2 Megabases (2 Mb) in size. Using two alternating electric fields, the system was named orthogonal-field alternating gel electrophoresis or OFAGE. DNA molecules have to reorientate at every change of the electric field. The reorientation time depends on the size of the DNA molecule allowing separation of intact chromosomes. Since 1984, a number of pulsed-field gel electrophoresis (PFGE) systems were developed and chromosomes with a size of up to 10 Mb (Orbach et al. 1988) were successfully separated.