Figure 3

Demonstration of the evaluation of Ecotilling signals using Vector NTI. This image shows a section of an Ecotilling gel targeting the 5' gene region of JUNB and showing two different signals. Each lane contains pooled PCR fragments of four different individuals. The DNA extracts were normalized and pooled prior to PCR amplification. Upper panel: A signal appears in two lanes at approximately 370 bp from the forward primer. The measured distances are reported in the lower right corner of the Vector NTI window. By looking up the respective position in Vector NTI shows that two adjacent SNPs are known at that position (consider the zoomed sequence) and a potentially regulatory region (shaded arrow) is located nearby downstream. Lower panel: A signal is located at approximately 245 bp from the forward primer. Inspection of the respective region in the Vector NTI data set reveals that no so-far known SNP is located at the respective region (considering that no SNP is annotated to the sequence). The signal is located immediately upstream of the exon (orange bar) and near a potentially regulatory region (shaded arrow and purple annotation). A reverse sequencing primer is located nearby downstream (bold blue arrow and green annotations). This procedure is shown in more detail by the additional file 3 (movie-2.avi).