Figure 3.

Dye terminator sequencing (BigDye 3.1 kit) of an ODN containing d^MeisoC and disoG used to optimize concentrations of d^MeisoCTP and disoGTP. The template sequence in the region shown is 3′-GCTGCTTCGTGCiGTiGAACiCATGiCCGCiGAiC TGATTTTTCiGTiGAACiCATGiCCGCiGAiCTGACATCTA-5′. Changing the concentrations of d^MeisoCTP and disoGTP caused differences in dye terminator signals at template disoG (black arrow) and d^MeisoC (red arrow) positions. With increasing concentrations of d^MeisoCTP, signals from ddT were suppressed opposite template disoG positions. With increasing concentrations of disoGTP, signals from ddA were suppressed opposite d^MeisoC template positions. These changes are indicative of competition for incorporation at non-natural template positions between a non-natural nucleotide and a specific natural nucleotide. (A) Sequencing with 100 μM d^MeisoCTP and 400 μM disoGTP largely suppressed terminator incorporation opposite the non-natural positions. Modest signal attenuation caused by unwanted strand termination was apparent. (B) As the concentrations of disoGTP and d^MeisoCTP were decreased (10 μM d^MeisoCTP and 10 μM disoGTP), ddA terminators were incorporated opposite template d^MeisoC positions and ddT terminators were incorporated opposite template disoG positions. Signal attenuation caused by unwanted strand termination was increased. (C) In the absence of disoGTP and d^MeisoCTP, more ddA was incorporated opposite template disoC and more ddT was incorporated opposite template d^MeisoG. Forced misincorporation at all non-natural template positions led to premature termination of the sequencing reaction, unlike in (A) and (B) where it was possible to incorporate some non-natural nucleotide. Similar reactions were performed using the BigDye 3.0 kit (Supplementary Figures S4 and S5).