Skip to main content
Figure 2 | BMC Biotechnology

Figure 2

From: Enhanced annealing of mismatched oligonucleotides using a novel melting curve assay allows efficient in vitro discrimination and restriction of a single nucleotide polymorphism

Figure 2

S1 nuclease treatment of wild-type and mutant templates annealed to mismatch-forming oligonucleotides. Oligonucleotides annealed at 20°C were incubated with 0.1 U/μl S1 nuclease at 20°C for 90 minutes. (A) Typical polyacrylamide gel electrophoresis depiction of uncut and nuclease-treated annealed oligonucleotides. Ladder: Low Molecular Weight DNA Ladder (NEB) (B) Comparative analysis of nuclease sensitivity against mismatch-inducing oligonucleotides annealed to wild-type (light grey) and mutant (dark grey) templates. One-way ANOVA was used to assess significance of degradation of the wild-type templates as compared to complimentary sequences. Two-way ANOVA was used to assess discrimination between wild-type and mutant templates for each mismatch set (* = p < 0.05; *** = p < 0.001). (C) S1 nuclease cleavage kinetics on stepwise increasing mismatch 'bubble' templates. Annealed oligonucleotides were incubated with 0.1 U/μl S1 nuclease, and incubated at 20°C for the indicated time. Reactions were stopped by adding 1 μl of 0.5 M EDTA and kept on ice prior to electrophoresis. Data is represented as mean percentage of DNA band intensity compared to uncut template ± SEM (N = 3).

Back to article page

BMC Biotechnology

ISSN: 1472-6750

Contact us