Genetic Sequencing troubles

[jhassett]

Hi all,

I am conducting genetic sequencing on the COI-COII region of the mtDNA of Apis mellifera mellifera but I am having trouble obtaining a sufficient quality genetic sequence, I can only get 70-80% coverage of the region.

DNA extraction by chelex and amplification by PuReTaq Ready-To-Go PCR beads provide adequate level of DNA for sequencing, also any purification step tried has yielded worse results. The region of interest if heavily A and T populated but steps have been taken to prevent this hindering the sequencing.

If anyone has encountered this problem before or has any advice it would be greatly appreciated.

Thanks,
Jack

[cascoamarillo]

Let's see,
First: "genetic sequencing", I guess that means Sanger sequencing, probably sequenced by capillarity (like ABI systems), right?
Second: "sufficient quality genetic sequence". Do you have clear and bright PCR bands? Never tried PuReTaq Ready-To-Go PCR beads before. What other purification steps have you tried?

Metazoan COI-COII genes shouldn't be hard to amplify and sequence (unless you encounter any 'numts' in you organism). Are you amplifying the entire region or using different primer pairs?

[jhassett]

Yes I am Sanger sequencing on an ABI 3130. I also have clear and bright PCR bands. I have tried the GENEJET purification kit and an in-house agarose gel purification method.
I am amplifying the the entire region using a single pair of primers

[cascoamarillo]

Good.
One more question; by "70-80% coverage of the region" you means... you sequence with one primer, or both and you get a bad coverage of a specific region when you combine them?

[jhassett]

I sequenced separately forward and reverse and combined the resulting sequences, at times the forward works much better and the reverse barley works and then at other times the opposite happens. rarely are they both good reads.

[cascoamarillo]

I guess this kind of hands-on troubles are hard to solve through a forum. It's difficult if I don't see the pcr gel, electropherogram, etc.
What I would try is to clone the fragment (a few ones) and sequence with sequencing vector primers. Let's see if there's something in the region (AT rich zones) which gives troubles. You can also try using different primers for sequence the region; not just the PCR primers and maybe try to avoid the AT areas. Do you need the entire fragment? How big is it? Maybe it is worthy to split it into two. It is for a phylogenetic study?
One method that really work for me in direct PCR sequence is ExoSapIT; you can work with small amounts of PCR product and gives you good reads.
Good luck