Figure 1.

SV40-based in vitro replication system of linear DNA with a stretch of T₂AG₃ repeats at one end. In vitro replication of linear DNA containing telomeric repeats at one end. pT2AG3 contains ∼1.9 kb telomeric repeats and a single restriction site of BbsI abutting the tract of telomeric repeats. BbsI leaves 4 nt 5′ protrusions at the digested sites and the 3′- recessive ends were filled-in with dATPs, dGTPs, biotinylated dUTPs and biotinylated dCTPs. Those biotin-labeled DNAs were captured on avidin-coated beads and subjected to replication reactions. After the reactions, bead-bound DNAs were collected and subjected to analysis. Only the replication products produced by a single round of replication from the original template DNAs retain the terminal biotin labels. Therefore, it is possible to analyze those products by analyzing the bead-bound DNAs. Note that one end of linearized pT2AG3 has a telomeric sequence to the extreme end in the same direction as that of the native telomeres (3′-OH of G-strand at termini). The BbsI recognition sequence is shadowed. Biotin-labeled nucleotides are shown by small filled circles.