FIG. 3.

LAM-PCR validation and sensitivity using Detroit 6 cells. Southern blot hybridization was performed on LAM-PCR products to determine assay sensitivity. Naïve human total cellular DNA (1 μg), containing various spiked copies of Detroit 6 cell DNA, was used as the template for LAM-PCR using an AAV cap-specific primer and a ligated blunt linker. Positive Southern hybridization (using an AAVS1 probe) detected amplification of as few as 160 Detroit 6 viral-cellular junctions. Shown in the bottom panel is an ethidium bromide stained gel of a human erythropoietin (cap) LAM-PCR genomic fragment that was generated using the same DNA templates used for the AAV LAM-PCR analysis. All samples amplified the expected 500-bp cap genomic fragment following LAM-PCR with an cap gene-specific primer (see Materials and Methods), confirming the DNA integrity of the sample and the reproducibility of the LAM-PCR assay.