- The signal terminates suddenly.
- The signal value is not bad at the beginning, but the peaks decline suddenly and the signal value drops, accompanied by some noise.
- Especially before the signal terminates, G/C rich regions appear in the template DNA.
There appear secondary construct at the area where the signal terminates, including the GC Rich region, Hairpin construct, siRNA constructs, and repetitive dinucleotides (such as GT, CT). Such area is folded as a hairpin structure, so that the Taq polymerase can not smoothly pass through this area.
- Use secondary constructed reagent to make sequencing. During the sequencing reaction, add 7-deaza-deoxy guanosine triphosphate (7-deaza-dGTP) to help the polymerase pass through the GC-rich region, or add DMSO, Betaine and etc. It’s effective in unfastening the secondary construct to help you solve the difficult sequencing.
- Use the primer at the other end to make sequencing.