Sequences always stop at secondary structure sites

Dear all,

I had some problems when I tried to sequence the ITS regions in ribosomal nuclear. They are the GC% high content regions. I tried to change the temperature regimes of sequencing reaction (to 98oc for 5 minutes in the begining of reaction), using DMSO (up to 10%) and designing new primers for this regions, but the results were not successful.

In my case, many sequences stopped at positions of 160-190bp (after poly-G area) or the bad signal (look like mistemplate) after this area.

Please help me to solve this problem if you had experience or met the same problems.

Thank a lots for your reading.

This is a hard problem to solve. On solution is to try using betaine in the sequencing mix (you will need to try a concentration range - say from 0.1M to 1.5M).

Another thing that works quite well if you are PCR amplifying your template before sequencing is to use 7-deaza-guanidine instead of dGTP in the nucleotide mix. 7-deaza-GTP disrupts the template secondary structure and allows the sequencing polymerase to read through the poly G region. The only thing to watch for is 7-deaza-G stops EtBr from fluorescing so that it will look like the PCR is very inefficient. This effect caught me out years ago when I was an undergraduate.

Daniel Tillett
Nucleics Support