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. 2004 Oct 28;32(19):e148. doi: 10.1093/nar/gnh150

Figure 2.

Figure 2

Improvement of signal-to-noise ratio in the EndoV/ligase assay when combining both enzymes in a single incubation step. Universal PCR primers (VicUniEV1F/NedUniEV2R), along with gene-specific primers F173 and R174, were used to amplify 350 bp fragments of p53 exon 8 carrying R273H (G→A) mutation. VIC- and NED-labeled mutation cleavage products are 158 nt (green arrow) and 195 nt (blue arrow) in size. The ‘split label, denaturation, renaturation’ method was used to generate heteroduplexes of either top strand wild-type:bottom strand mutant (G/T mismatch) or top strand mutant:bottom strand wild-type (A/C mismatch). Wild-type PCR product mixtures were used as controls (G:C match). Tested EndoV/ligase assay conditions are briefly indicated on top and bottom of the figure and detailed in Table 2. Condition III is a standard two-step reaction, while condition IV combines both enzymes in the first incubation step [EndoV + ligase], followed by a second incubation in ligase buffer. Reaction mixtures were electrophoresed on the ABI 3730 fluorescence-based capillary electrophoresis instrument. Data were analyzed using Gene Mapper fragment analysis software.