nucleotide mutations introduced during transformation - (Feb/12/2008 )

Dear Sirs,
I really need sombody to help me.

I am constructing retroviral vectors: I amplify the fragment of interest, dou ble digest, ligate, transform, perform mini-preps and sequentiate.

All my sequence presents nucleotide mutations not expected.

I sequence the PCR prducts before ligation and see not mutations so I discarted errors introduced by Taq pol.

Furthermore, all clones from the same transformation (aprox 10 clones) present mutations at different positions.

In addition, if I sequence the same prep different days I get the same sequence so I also discard errors introduced during sequence reaction.

What should I do?
I will be very pleased if someone could help me.

best wishes
Laura

This is not unexpected. When you sequence a PCR product, you sequence a large pool of molecules, each of which may be different. If they differ in many different locations you will not notice the effect since ON AVERAGE the sequence is correct at any specific location. When you clone, you choose exactly one molecule from the PCR pool. This molecule may have specific errors (different from the errors of other PCR products). You amplify this sequence from one molecule when you clone it, to very large numbers when you prep plasmid DNA. The sequenced products will now show the original error, as will all resequencing of that clone. Different clones will in general have different sequences.

The solution is to (1) amplify the initial template DNA less, which will reduce the error probability. You can do this by adding more template initially, and fewer cycles. (2) use a low error rate PCR enzyme, such a Phusion.

My advice is to use a proof reading polymerase ( KodHifi, Phusion etc) when doing cloning work. Do not use Taq to make your cloning fragment. Taq has a high error rate and thus makes mistakes rather often.

For cloning work, I do not exceed 25 cycles when conducting PCR.

Dears phage 434 and perneseblue,
thank you very much for your advice!!!
I will beggin right now a new PCR with high fidelity
all my best wishes
Laura