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. 2019 May 4;47(13):6769–6782. doi: 10.1093/nar/gkz335

Figure 2.

Figure 2.

TLS-associated nucleotide misincorporation on UVB-irradiated ssDNA template. (A) Illustration of a synthetic DNA substrate used to quantify TLS-mediated nucleotide misincorporation. The NGS-primer contained an A-adaptor (red box) and a barcode (yellow box, BC) while the template contained a P1-adaptor (green box) for Ion Torrent NGS. Only the primer extension products that contain both adaptors are sequenced, reading a 73-nt region (position #1–73), in which 20nt (#1–20) belong to the primer and 53nt (#21–73; variable region) are produced by primer extension. The NGS primer had a one-base mismatch at position #10 (10G) to distinguish the primer-extension products from template-extension products. (B) The ssDNA template was irradiated with the indicated dose of UVB, then hybridized with NGS primer containing a unique barcode and subjected to primer extension reaction first by yPol δ and then by premixed yPol η + yPol ζ. Full-length products were directly analysed by NGS, and single nucleotide misincorporation frequencies per full-length product are indicated by bars (green:T; yellow: G; blue: C; and red: A).