Problems with Mutations of 2.1 kb Gene - (Oct/16/2008 )

Hallo,

i know this questions was asked serveral times, but the answeres couldn't help me so far.

I'm trying to clone a 2,1 kb gene from Brain cDNA. Everythings seems to work fine, but the sequence of my Teasy clones have around 2-3 mutations (different mutations each clone). The spectra quality of the sequencing is high.

I tried Platinium Taq, Invitrogen High Fidelity und Pfu (with Pfu I could not get a PCR product so far).
Number of mutations don't seem to vary by changing from Taq to Hifi.

What could create mutations beside the type of polymerase?
What strategy would you recommend?

Thank you very much for your help!

Cheers,
Brix

Perhaps it is not a problem of fidelity. Are you sure that the "mutations" are introduced by the taq and they are not polymorphism??
try to check that information in ncbi, snp. or?..try to amplify the same gene of adn of differents samples and see if polymorphism
exist.

tell me afterward what happen!!! cause i always reply but i dont know what happen!!

I use Phusion - it is the best - great robustness and rare infidelity. Apart from that, you've got a couple of options:

1) sequence more clones. They may be polymerase errors but they might just be polymorphisms too. All because they aren't in the literature or not on NCBI as an SNP doesn't mean it isn't a polymorphism. Some genes just seem to have a lot of variation.

2) Mutate one of your clones back to the original sequence.

I would try option 1 on a few more clones and if it doesn't work out, then go for option 2. Or you can do option 1 and 2 simultaneously.

Good luck,
Rob

Another possibility is that your clone is toxic, and the correct clone is strongly selected against. Are the mutations you see frameshifting or truncating the gene?

Yes, I have seen many inactivating mutations when trying to clone into vectors containing the ccdB lethal gene in the (parent) cloning site. Most people ignore these when cloning since they are interested only in the clones that worked. We try to identify what went wrong, and sequence the failures. Many of the failures are parent vector with mutations which inactivate the lethal gene through frame shifts or early stops.

Hi there
Sorry to hear about your mutations! How many cycles of PCR are you doing?
Clare