Methods to Study Repeat Fragility and Instability in Saccharomyces cerevisiae

Trinucleotide repeats are common in the human genome and can undergo changes in repeat length. Expanded CAG repeats have been linked to over 14 human diseases and are considered hotspots for breakage and genomic rearrangement. Here, we describe two Saccharomyces cerevisiae based assays that evaluate the rate of chromosome breakage that occurs within a repeat tract (fragility), and a PCR-based assay to evaluate tract length changes (instability). The first fragility assay utilizes end-loss and subsequent telomere addition as the main mode of repair of a yeast artificial chromosome (YAC). The second fragility assay relies on the fact that a chromosomal break stimulates recombination-mediated repair. In addition to understanding the role of fragility at repetitive DNA sequences, both assays can be modified to evaluate instability of a CAG repeat using a PCR-based assay. All three assays have been essential in understanding the genetic mechanisms that cause chromosome breaks and tract-length changes at unstable repeats.