Tracking Expansions of Stable and Threshold Length Trinucleotide Repeat Tracts In Vivo and In Vitro Using Saccharomyces cerevisiae

  • Gregory M. Williams
  • Athena K. Petrides
  • Lata Balakrishnan
  • Jennifer A. SurteesEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2056)


Trinucleotide repeat (TNR) tracts are inherently unstable during DNA replication, leading to repeat expansions and/or contractions. Expanded tracts are the cause of over 40 neurodegenerative and neuromuscular diseases. In this chapter, we focus on the (CAG)n and (CTG)n repeat sequences that, when expanded, lead to Huntington’s disease (HD) and myotonic dystrophy type 1 (DM1), respectively, as well as a number of other neurodegenerative diseases. TNR tracts in most individuals are relatively small and stable in terms of length. However, TNR tracts become increasingly prone to expansion as tract length increases, eventually leading to very long tracts that disrupt coding (e.g. HD) or noncoding (e.g., DM1) regions of the genome. It is important to understand the early stages in TNR expansions, that is, the transition from small, stable lengths to susceptible threshold lengths. We describe PCR-based in vivo assays, using the model system Saccharomyces cerevisiae, to determine and characterize the dynamic behavior of TNR tracts in the stable and threshold ranges. We also describe a simple in vitro system to assess tract dynamics during 5′ single-stranded DNA (ssDNA) flap processing and to assess the role of different DNA metabolism proteins in these dynamics. These assays can ultimately be used to determine factors that influence the early stages of TNR tract expansion.


Trinucleotide repeat Expansion Contraction Polymerase chain reaction Saccharomyces cerevisiae DNA replication Repeat tract dynamics Microsatellite instability 



We are particularly grateful for the generosity of Dr. Robert Lahue in providing reagents and for many useful discussions and comments in developing this protocol. We are also grateful for discussions with Dr. Catherine Freudenreich, Dr. Robert Bambara, and Dr. Eric Alani. Work in the Balakrishnan laboratory is supported by the National Institutes of Health (GM0938328 to L.B.) Work in the Surtees laboratory is supported by the National Institutes of Health (GM087459 to J.A.S.) and the American Cancer Society (RSG-14-235-01 to J.A.S.). J.A.S. is an ACS Research Scholar.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Gregory M. Williams
    • 1
    • 2
  • Athena K. Petrides
    • 3
  • Lata Balakrishnan
    • 4
  • Jennifer A. Surtees
    • 5
    • 6
    Email author
  1. 1.Centre for Chromosome BiologyNational University of IrelandGalwayIreland
  2. 2.Galway Neuroscience CentreNational Universityof IrelandGalwayIreland
  3. 3.Department of PathologyHarvard Medical SchoolBostonUSA
  4. 4.Department of BiologyIndiana University Purdue University IndianapolisIndianapolisUSA
  5. 5.Department of Biochemistry, JacobsSchool of Medicine and BiomedicalSciencesState University of New York atBuffaloBuffaloUSA
  6. 6.Genetics, Genomics and Bioinformatics Program, Jacobs School of Medicine and Biomedical SciencesState University of New York at BuffaloBuffaloUSA

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