Genomic DNA PCR and sequencing confirm precise monoallelic gene correction and Cre-LoxP excision. (A) Schematic of genomic DNA PCR using primers in 5′-untranslated region of exon 1 and exon 2 of HBB. With a short 72°C extension step (15 seconds), only the mutant allele (291 bp) and the corrected allele after excision (337 bp) can be amplified. (B) DNA gel shows 1 band for the c36 clone representing the mutant allele that can be amplified by the PCR protocol, and 2 bands from every cre clone representing both mutant allele and corrected allele after excision. (C) The mixed PCR products from cre4 iPSCs (after gene correction and Cre-LoxP excision) were cloned into a TOPO vector, and individual clones were sequenced. Among 8 sequenced clones, 4 clones (50%) were shown to contain a mutant allele (top panel), and 4 clones (50%) bore the corrected allele with a remnant loxP site after excision (bottom panel).