Detection of Chimerism in YAC Clones

Chimerism, the presence of noncontiguous DNA fragments in the same clone, is one of the most common problems encountered when working with yeast artificial chromosomes (YACs). Its frequency can vary among the different libraries, but on average, 40–60% of YAC clones among the most used libraries are chimeric (1 ). To determine if a given YAC clone is chimeric, the most obvious approach is to isolate both ends of the insert and map them to assess if they have the same chromosomal origin, However, this approach could be time consuming, especially when an investigator deals with many YACs isolated from the same genomic locus and wants to identify very quickly the clones to be characterized. An alternative approach involves the fluorescent in situ hybridization (FISH) of Alu polymerase chain reaction (Alu -PCR) products, from YAC DNA to human metaphase chromosomes. This method, although very powerful, has the disadvantages of requiring expertise in preparing metaphase chromosomal spreads, and the need for specialized equipment.