ABRUPT SIGNAL LOSS

1) Too Much Template: Because the majority of the dye is labeled during the first few rounds of cycle sequencing, the smaller fragments generated by the reaction are resolved well. However, as the size increases there is a dramatic drop in peak height resulting in a shorter than expected sequence read. Typically, the initial 100 bases are off scale. Solutions: Verify the concentration of DNA template. Samples that display this phenomenon are automatically diluted and re-run.

2) Secondary Structure: The sequence of the DNA inherently forms a hairpin loop, restricting the activity of the sequencing enzyme. Solutions: Request that your DNA be sequenced under GC rich conditions. The additional amount of enzyme and increased denaturation temperature used in this protocol may help overcome secondary structure.

3)GC Rich: DNA with a high GC content can fold onto itself in the form of hairpin loops, decreasing the efficiency of the modified Taq polymerase. Solutions: Request that your DNA be sequenced under GC rich conditions. The additional amount of enzyme and increased denaturation temperature can sometimes be enough to overcome the hairpin loops.