Figure 4.

Dye terminator sequencing of two ODNs used to examine d^MeisoC and disoG in all possible natural nearest-neighbor contexts (Supplementary Figures S6 and S7 contain additional sequences). An ODN containing four d^MeisoC positions (red arrows) was sequenced in a BigDye 3.1 reaction in the presence of (A) 300 μM disoGTP and 0 μM d^MeisoCTP or (B) 0 μM disoGTP and 0 μM d^MeisoCTP. The template sequence in the region shown is 3′-TGCTGCTGAAAGCiCATGTCAGCiCCTGTCAGCiCGTGTCAGCiCTTTGTCAG-5′. Very large ddA terminator signals were always observed opposite d^MeisoC in the absence of disoGTP, perhaps indicating a transition state base pairing geometry different from complementary pairings. An ODN containing four disoG positions (black arrows) was sequenced in a BigDye 3.1 sequencing reaction in the presence of (C) 100 μM d^MeisoCTP and 0 μM disoGTP or (D) 0 μM d^MeisoCTP and 0 μM disoGTP. The template sequence in the region shown is 3′-TGCTGCTGAAAGAiG ATGTCAGAiGCTGTCAGAiGGTGTCAGAiGTTGTCAG-5′. Terminator ddT was always incorporated opposite disoG in the absence of d^MeisoCTP. Extremely large terminator signals were observed at the position following disoG for all conditions. Additionally, a noticeable signal that may correspond to the polymerase skipping over a fraction of template disoG positions was visible opposite disoG. Little, if any, signal attenuation caused by unwanted strand termination was visible upon encountering isolated d^MeisoC or disoG positions, either in the presence or absence of complements disoGTP and d^MeisoCTP. This contrasts with Figure 3C, in which several proximate non-natural template positions caused premature termination of sequencing reactions lacking disoGTP and d^MeisoCTP.