Figure 3

Detection of chromosome 15 AC216176 L1Hs by the 3’ junction ddPCR assay. The L1Hs 3’ junction ddPCR assay uses a L1-specific primer, L1-specific 5’-FAM labeled Taqman™ probe, and a locus-specific primer near the Chromosome 15 AC216176 3’-insertion junction, as shown in Figure 1B. The FAM fluorescent signal (Ch 1) for each droplet is plotted on the y-axis for each of the ddPCR experiments, which are separated by a dotted yellow line, with input DNA indicated above each experiment. Each droplet is cumulatively counted as an ‘Event Number’ for the ddPCR experiments analyzed in tandem, and plotted along the x-axis. The positive droplet fluorescence threshold is indicated by the magenta line, which determines whether a droplet is considered positive or negative for FAM fluorescence. Thus, the blue dots represent individual droplets that contain at least one copy of the L1 locus tested. We tested 200 ng of BamHI-digested genomic DNA from HeLa cells, which contain the polymorphic L1 element, and tenfold dilutions of this same sample as a mixture with BamHI-digested genomic DNA from HEK293 cells, which do not have this polymorphic L1 insertion. Percentages given reflect the amount of input DNA with 100% corresponding to 200 ng of DNA. This assay robustly detects the 3’-insertion junction of the polymorphic full-length AC216176 L1Hs element when present in the genomic DNA from a cell line positive for that polymorphism (HeLa 100%), but not in a cell line negative for that polymorphism (HEK293 100%). L1-positive droplets are observed at dilutions as low as 0.01% of the DNA with this assay.