Abstract
Approximately 3% of the human genome is composed of short tandem repeat (STR) DNA sequence known as microsatellites, which can be found in both coding and non-coding regions. When associated with genic regions, expansion of microsatellite repeats beyond a critical threshold causes dozens of neurological repeat expansion disorders. To better understand the molecular pathology of repeat expansion disorders, precise cloning of microsatellite repeat sequence and expansion size is highly valuable. Unfortunately, cloning repeat expansions is often challenging and presents a significant bottleneck to practical investigation. Here, we describe a clear method for seamless and systematic cloning of practically any microsatellite repeat expansion. We use cloning and expansion of GGGGCC repeats, which are the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as an example. We employ a recursive directional ligation (RDL) technique to build multiple GGGGCC repeat-containing vectors. We describe methods to validate repeat expansion cloning, including diagnostic restriction digestion, PCR across the repeat, and next-generation long-read MinION nanopore sequencing. Validated cloning of microsatellite repeats beyond the critical expansion threshold can facilitate step-by-step characterization of disease mechanisms at the cellular and molecular level.
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The datasets generated and analyzed during this study will be made available on a public database to be determined or by request to the corresponding author.
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Acknowledgements
We thank T.R. Murphy of the Hamilton-Brehm laboratory (SIU) for assistance in MinION data analysis.
Funding
This work was supported by an ALS Association grant to K.T.G., a Judith and Jean Pape Adams Charitable Foundation grant to K.T.G., and a Department of Defense ALSRP grant to K.T.G.
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KJR designed and executed RDL cloning experiments, performed data analyses, and wrote the manuscript. KNO designed and executed RDL cloning experiments, performed data analyses, and assisted in manuscript preparation. AAP performed and analyzed MinION sequencing, developed the bioinformatics workflow, and assisted in manuscript preparation. MB executed RDL cloning experiments. AJH assisted with RDL cloning and PCR experiments. KTG designed, planned and supervised all experiments, interpreted results, and wrote the manuscript.
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Rohilla, K.J., Ovington, K.N., Pater, A.A. et al. Systematic microsatellite repeat expansion cloning and validation. Hum Genet 139, 1233–1246 (2020). https://doi.org/10.1007/s00439-020-02165-z
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DOI: https://doi.org/10.1007/s00439-020-02165-z