Quantitative PCR assay. (A) Graphical representation of primer locations relative to the genomic structure of wild-type and transgenic knockout SV129 mice. Primers A, B, and C were used for PCR amplification, whereas primer D is for Southern blotting. The border for the knockout is the EcoRI restriction site. The lower panel shows a typical PCR reaction using primers A, B, and C in FACKO mutants (Mut) and heterozgyotes (Het). The wild-type DNA yields a 463-bp PCR product, and the mutant gives a 292-bp PCR product. (B) Ethidium bromide-stained gel of standard curve for quantifying DNA contribution. Wild-type and knockout DNA was mixed in various ratios then subjected to PCR. This gel would then be Southern-blotted and quantitated as described.