Dear experts,
I need your assistance regarding the selection of the TA clones. There are some mutations/changing in some nucleotide like A is replaced with C, T replaced with G etc at 2-3 different position. My question is that, does this change will affect my end result if I use these clones (most similar ones to my ref seq) for next step (construction of over-expression vector)?
These mutations are exist in the form of amino acids. What are the possible reasons for these mutations? And how does it effect the end result?
I guess you are talking about PCR products cloned in a TA-cloning vector.
For the possible reasons: There may be a large sequence variation in your research organism, a low quality of your template DNA combined with too many amplification steps and then there is the choice of your polymerase.
The databases may give you an idea about SNPs in those positions. In general, I would recommend use a proofreading enzyme like NEB Q5-Polymerase. You should not proceed in case you have a mutated protein. But even silent mutations may have negative effects on over-expression.
If your goal is to express the gene in order to produce a recombinant protein, you should definitely discard the clones harbouring the spurious mutations, unless they fail to affect the AA sequence of the encoded polypeptide. The problem is that the Taq polymerase that you use to PCR your gene has poor fidelity. Thus, I would suggest that you use a high fidelity polymerase such as Phusion or Q5, isolate the ampli on and then treat it with Taq polymerase + dATP to add the A overhang that you need for TA cloning. But if you want to use the gene for recombinant protein production, why don’t you amplify it with primers bearing adequate restriction sites and clone it directly into you expression vector ?
Dear Uwe Borgmeyer Pierre Béguin Many thanks for your reply. I am looking to find out the best clone to construct over-expression vector. First I did PCR, after getting the exact size of amplified band. I purified the PCR product (40μL) using kit. After the TA cloning, I cultured the 5-alpha cells on LB plate. Then I selected seven clones from each plate and send them for sequencing. Now I got the sequence of TA clones with some mutations. So, now how I can select best clones to get their plasmid and move towards the vector construction? And how does these 1 or 2 nucleotide change will affect my end result?
Honestly, I would do the PCR again with a proofreading enzyme and as Pierre Béguin suggested use primers bearing adequate restriction sites. If you later publish your data you want to avoid a discussion about some mutations.
If you want to go ahead with your clones, discard those giving rise to an altered amino acid sequence. Then have to check the synonymous mutations using a codon frequency table for your target expression species (E. coli, ...) . Avoid those with a very low frequency.
That minimizes the risk of unwanted effects. If all you want to do some overexpression, you are fine.
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