Figure 3.

A, nested PCR-based incorporation assay for DNA containing Z:P nucleobase pairs using a target DNA sequence (cyan-red-green) composed of only WC base pairs. Replication is initiated by chimeric primers that can bind to the original gene but have UBPs at the 5′-end. These chimeric primers are present at low concentration (Step 1). After consumption of the chimeric primers (Step 2), amplification is continued by external 5′-PPPP-OH primers (containing only UBPs), which are present at high concentration. As a result, full-length amplicons are produced only if the DNA polymerase can efficiently incorporate UBPs (based on Fig. 2 of Ref. 65). B, PCR-based replication fidelity assay for DNA containing Z:P nucleobase pairs. If the UBP is lost to a T:A base pair, then a Dra restriction site (red) is generated in the amplicons. Similarly, if the UBP is lost to a C:G base pair, then an Alu restriction site (blue) is produced. C, gel showing misincorporation rates in the PCR amplification of template DNA in the presence of dZTP and/or dPTP components using WT Klentaq and WT Phusion DNA polymerases at different pH values (64). Four parallel PCRs were performed followed by digestion with PspOMI for 16 h. The ratio between the amount of full-length product (FLP) and all oligonucleotides gives the misincorporation rates shown in the figure. Lane 1, negative control PCR amplification of template DNA in the presence of WC dNTPs (200 mm each), followed by digestion with PspOMI. Lane 2, five-letter PCR amplification of template DNA in the presence of WC dNTPs (200 mm each) and dZTP (25 mm), followed by digestion with PspOMI. Lane 3, five-letter PCR amplification of template DNA in the presence of WC dNTPs (200 mm each) and dPTP (25 mm), followed by digestion with PspOMI. Lane 4, six-letter PCR amplification of template DNA in the presence of WC dNTPs (200 mm each), dZTP (25 mm), and dPTP (25 mm), followed by digestion with PspOMI. Taken from Ref. 64 and used with permission.