Primer extension and melt analysis. On the left, the diagrams show where the 3′-end of primer hybridization to the template ended (after the iG in controls or before the iG for experimental). On the right, melt analysis of primer extension products where bases opposite iG were controlled (X = A,G,C,T,iC) or experimentally determined (Y) is shown. All reactions were treated with TiTaq, dG,dA,dT,dCTP, quencher-modified diGTP and diCTP when designated. R, tetrachlorofluorescein; ∗, Dabcyl.

Acid cleavage analysis. (A) Acid cleavage products using natural (odd lanes) and non-natural (even lanes) DNA targets. Reactions were resolved by PAGE and detected by fluorescent imaging. Lanes 1 and 2 were loaded with extension reactions that were initiated with 10¹⁰ copies of input target. Lanes 3 and 4, 5 and 6, 7 and 8 and 9 and 10 were loaded with PCR reactions initiated with 1.5 × 10¹–10¹⁰ input targets in 1000-fold increments and amplified for 40 cycles. Bands that appear in the scan that were used to determine percent cleaved are labeled (FLP, full length product; CP, cleavage product). (B) Cleavage analysis. Using a series of 10-fold dilutions of the non-natural template and 40 cycles of PCR, the percent of cleaved product was determined as above and plotted against input copy number.

Molecular beacon analysis. Amplification analysis using reactions containing Template 1 (A) or Template 2 (B) and a molecular beacon specific for Template 1. Ten-fold dilutions of each target from 10² to 10⁷ were constructed and added to the reaction series. The reactions were fluorescently monitored through 40 cycles of PCR.