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Fig. 5 | Molecular Neurodegeneration

Fig. 5

From: Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease

Fig. 5

Characterization of the affected C9orf72 repeat expansion carriers using standard methodologies. a, d We first performed fluorescent PCR to determine the individuals' non-pathogenic repeat sizes. Genomic DNA was PCR-amplified with genotyping primers and one fluorescently labeled primer. Fragment length analysis of the PCR product was then performed on an ABI3730 DNA analyzer and visualized using GeneMapper software. A peak is observable at 129 bp (a) and 165 bp (d), indicating that the non-pathogenic alleles for samples 1 and 2 contain two and eight repeats, respectively. A single peak also indicates that the individual is either homozygous for the given allele, or also has an expansion. b, e To determine whether the individuals had a repeat expansion, we performed a repeat-primed PCR analysis. PCR products of a repeat-primed PCR were separated on an ABI3730 DNA analyzer and visualized by GeneMapper software, showing a stutter amplification characteristic for a C9orf72 repeat expansion. This does not indicate expansion size, however. c, e After determining the individuals were expansion carriers, we performed a Southern blot to estimate the size. The Southern blots reveal a long repeat expansion in other individuals for whom cerebellar tissue was available, including positive controls (POS CON; lanes 1–5, and 1 and 3, respectively) and our patients of interest (CASE; lanes six and two, respectively). DIG-labeled DNA Molecular Weight Markers (Roche) are shown to estimate the repeat expansion’s size. Measurements were based on multiple separate Southern blots for each case; for simplicity one representative Southern blot is shown. The most abundant expansion size in samples 1 and 2 are estimated around 1083 (8.8 kb) and 1933 repeats (13.9 kb), respectively. The smears ranged widely, demonstrating the heterogeneity (i.e., mosaicism) of this repeat expansion within a small piece of tissue. This demonstrates the importance of additional long-read sequencing studies to characterize the repeat at the nucleotide level

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