Systematic microsatellite repeat expansion cloning and validation
| Autor: | Kushal J. Rohilla, Keith T. Gagnon, Adrian A Pater, Maria Barton, Katy N Ovington, Anthony J Henke |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Genotype
Genetic Vectors Gene Expression Computational biology Biology Polymerase Chain Reaction Article DNA sequencing 03 medical and health sciences Microsatellite Repeat Escherichia coli Genetics Humans Cloning Molecular Genetics (clinical) 030304 developmental biology Cloning 0303 health sciences DNA Repeat Expansion Base Sequence C9orf72 Protein Genome Human Amyotrophic Lateral Sclerosis 030305 genetics & heredity High-Throughput Nucleotide Sequencing DNA Restriction Enzymes Recombinant Proteins Frontotemporal Dementia Microsatellite Restriction digest Human genome Nanopore sequencing Trinucleotide repeat expansion Microsatellite Repeats |
| Zdroj: | Hum Genet |
| ISSN: | 1432-1203 0340-6717 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink