My target gene contains two PstI sites. Is it possible to remove them through PCR? Do I need to run PCR twice or just once? My target gene was cloned into a vector, more specifically, it is a BioBrick.
Is your target gene already cloned into a vector? Then you can do site directed mutagenesis, which is done via PCR.
You can do site-directed mutagenesis on amplicons using adapted primers. You can use a similar approach to this (http://www.methods.info/Methods/Mutagenesis/PCR_splicing.html), but with altering the restriction sites instead of creating a deletion.
I would do site-directed mutagenesis (SDM). There are very reliable and simple kits that are commercially available for this. Change just one nucleotide on each undesired restriction site. Remember if those sites are in an open reading frame, make sure you are not changing the relevant codon to an amino acid with completely different (or otherwise undesirable) properties.
I would do mutagenesis. If your two sites are far, you can do the mutations in one step, otherwise if the primers of mutagenesis superimpose themselves you need two steps.
Do you have any other restriction sites around 1 of the pst1 sites? You could cut with PST1 and then blunt the parental vector and then reconstitue the vector with a PCR product which has a silent mutation in the PST1 site to remove it. As the PCR product goes in blunt ended it is easier to do 1 site, if you have another unique restriction site in the target. You could do both pst sites at once, but the PCR product can go in in either orientation then (if the gene is toxic you will predominantly select the backwards ones). It saves doing two site directed mutagenesis reactions though. Don't forget to kinase the primers first as you will be going in blunt.
While I think all the above work there is another possibility and it is dependent on the size of your DNA and the amount of money you wish to spend. Lately, for smaller fragments (
SDM is best way to go. There are protocols for one round PCR and two round PCR. I will mutate one site then I will do the second. There are very interesting method papers to backup both ideas.
Again if you design good primers, you could do both site at one time. I have seen examples where they removed a N-terminal His tag and inserted a C-terminal his tag in single PCR, and their Gel picture is convincing. So its possible.
Hi, Sophia, I agree all others' suggestion to remove the restriction sites use site-directed mutagenesis methods. However, you also can think about using two different restriction endonucleases, which produce compatible overhangs, to remove the restriction site. For your case, you want to remove two PstI site in plasmid. PstI recognition sequence is CTGCAG, another restriction enzyme NsiI, which recognition sequence is ATGCAT, PstI and NsiI, both produce the same 3′ overhang sequence. The digestion products from these two enzymes are exactly matching and can be ligated well. However, neither the PstI or NsiI sites are regenerated in the ligation. What you have to do is to synthesis two primers, which use to amplify the fragment between two PstI sits in your plasmid. It is important that you should replace PstI recognition sequence with NsiI sequence close to the 5’ end in both of the specific primers. Then do PCR, digest the PCR product (insert) and vector with NsiI and Pst I, respectively, purify the insert (PCR product) and vector (Plasmid), ligate them and do transformation… After that subcloning you will get a new construct which should be attained your target. Good luck!
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