Analysis of Yeast Artificial Chromosome Clones

The technology of cloning large fragments of DNA in yeast as yeast artificial chromosomes (YACs) (1 ), combined with that of electro-phoretic separation of large fragments by pulsed-field gel electrophoresis (PFGE), has revolutionized research in molecular genetics. In addition to the large insert size, the YAC cloning system offers relatively unbiased genome representation compared to the conventional bacterial cloning systems based on plasmids or bacteriophages (2 ,3 ). The enormous impact of YAC cloning and PFGE techniques on physical mapping is being realized with the building of multimegabase contigs (contiguous cloned stretches of DNA). Some examples include mapping and cloning of a large gene like cystic fibrosis (CF) (4 ), of a chromosomal band like that of 18q21.3 (5 ), of a substantial portion of an entire human chromosome, Xq24–28 (3 ), and even an entire organism, C. eI.egum (2 ). Similarly, the use of YAC clones in the identification of disease-related genes like neurofibromatosis (NFl) is well documented (6 ). PFGE techniques play a major and critical role in the construction (see Chapter 16 ) and characterization (later in this chapter) of YAC clones.