Extended Data Fig. 2: APP gencDNA detection by genomic DNA PCR, DISH, and targeted genomic pull-down.
From: Somatic APP gene recombination in Alzheimer’s disease and normal neurons
a, Duplicate gel from Fig. 2b, with more sensitive thresholds to show the clear absence of PSEN1 bands. b, Nested PCR was used with alternative APP primers (three total sets: APP 1–18, APP 1–18N, and APP 2–17). c, Cloning and Sanger sequencing of indicated bands (red numbers in b) revealed novel APP gencDNAs (see Fig. 1 for legend and nomenclature). d, APP 1–18 DNA PCR showed no products in non-neuronal cell types: IMR-90 (human lung fibroblast), HEK (human embryonic kidney) and non-neuronal (NeuN-negative) genomic brain DNA from individuals with and without SAD. RNaseP was used as a positive control. e, APP mRNA is expressed in HEK-293 and IMR-90 cells; 18S rRNA used as a positive control. f, Digestion with the off-target restriction enzyme XbaI did not affect DISH3/16 or DISH16/17 signals. g, h, Synthetic DNA containing 16/17 (g) or 3/16 (h) target sequences (target), or wild-type human genomic APP sequences lacking IEJs and exon–exon junctions (mutant target) were introduced by retroviral transduction into NIH-3T3 cells. DISH16/17 and DISH3/16 signals from both sense and antisense probes were detected only in target infected cells. i, Schematic of Agilent SureSelect targeted DNA pull-down. j, Agilent SureSelect hybridization enrichment targeted the entire genomic locus of APP and showed unbiased sequencing depth across the full genomic locus. Exons and introns are shown on two scales.
