Figure 3
PCR confirms absence of the HPV6AI1 site in the recombinant cosmid clone CosC2. (A) Gel electrophoresis of products amplified from human genomic DNA and CosC2 DNA using PCR primer sets A, B and C, and Alu-specific primers (refer Figure 3C, Table 2). In the left panel, lanes 1–3 show genomic DNA fragments amplified with primer sets A (615 bp product), B (535 bp product), and C (699 bp product), all at 1.5 mM Mg++ and annealed at 60°C. Lane 4 shows CosC2 DNA amplified with Alu primers A51/Sb2ins2. Lanes 5 and 6 are, respectively, negative (no DNA) and positive genomic DNA controls for this primer set. Lane N is a negative control (no DNA) for primer set A. In the right panel, lanes 1–3 are as in the left panel except that reactions were at 2 mM Mg++ with annealing at 52°C. Note the presence of additional bands in lane 3 under these relaxed stringency PCR conditions. Lanes 4–6, under the same conditions, show no product from the CosC2 template using the primers sets A, B and C respectively. In both left and right panels, M is a 100 bp molecular weight reference ladder. (B) Somatic cell hybrid panel showing Pst I fragments of ~9.5 kb detected in control male and female genomic DNA lanes, and in the chromosome 14 specific lane (arrow) after hybridization with the 32P-dCTP labeled 615 bp fragment amplified using primer set A. λ Hind III fragment sizes are indicated to the left of the panel. (C) Alignment of the published sequence X77607 with contig NT_026437.9 from chromosome (chr) 14. In the lower figure, the thin grey line corresponds to chr 14 sequence, and thicker solid (repeat-free) or hatched boxes correspond to regions that have overlapping homology with X77607. Repeat sequences identified using CENSOR are shown as diagonal hatched regions corresponding to CHESHIRE_B (narrow downward), LIPB4 (wide upward), L1PREC2 (narrow upward) and THE1B (wide downward). Flanking nt sequence locations are indicated for each contig, as is the location of the HPV6AI1 integration site. Locations of primer pairs A-C are marked by forward (f) and reverse (r) arrows. The bracketed region marks 209 bp of chr 14 sequence not present in X77607.