Systematic microsatellite repeat expansion cloning and validation
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ORIGINAL INVESTIGATION
Systematic microsatellite repeat expansion cloning and validation Kushal J. Rohilla1 · Katy N. Ovington1 · Adrian A. Pater2 · Maria Barton1 · Anthony J. Henke2 · Keith T. Gagnon1,2 Received: 7 January 2020 / Accepted: 4 April 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
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 GGGGCCrepeats, 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 GGGGCCrepeat-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.
Introduction The Human Genome Project was completed in April 2003 and resulted in sequence for the vast majority of the genome, including about 99% of genic regions. However, a surprising fraction of the genome still remains unsequenced or unmapped due to highly repetitive regions. These uncharacterized regions are primarily composed of repetitive DNA sequences including short tandem repeats (STRs), or microsatellites (Miga et al. 2015). Microsatellites consist of simple sequence motifs of one to six nucleotides repeated at least 5–15 times at a genetic locus (Ellegren 2004; Rohilla and Gagnon 2017). Microsatellites account for a significant source of genetic diversity and are the basis for DNA fingerprinting techniques (Jeffreys et al. 1985; Roewer 2013;
Weischenfeldt et al. 2013). With respect to disease, STRs can be unstable and undergo expansion that leads to pathogenicity in succeeding generations (Brouwer et al. 2009; Paulson 2018). These expansions are the leading cause of a growing list of more than 30 neurological disorders including Huntington’s disease (HD), numerous spinocerebellar ataxias (SCA disorders), fragile X syndrome (FXS) and fragile X-associated tremor/ataxia syndrome (FXTAS), myotonic dystrophy type 1 (DM1) and type 2 (DM2), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD
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