how to remove a restricted endonuclease site - (Mar/16/2009 )
I have to remove a restricted endonuclease (RE) site in a protein, which belongs to pst I. The recognized sequence is CTGCA G. Small gap in the sequence indicates the digestion site. And CTG and CAG encode amino acids, respectively. My idea is change CAG to CAA, both codon encodes Gln. If idealy, it should be remove RE site without changing amino acid sequence in the protein.
My question is whether this point mutation could really eliminate the RE recognization and digestion. Someone claimed they perform point mutation in RE sites, however, protein still is digested by RE. Whether the mutated site design has some tricks I don't kown. Any people any experiences were welcome.
For PstI, a single bp change to its restriction site would be sufficient to knock out the site.
This strategy will work fine. But the DNA is not a protein(!).
perneseblue on Mar 17 2009, 10:30 AM said:
phage434 on Mar 17 2009, 10:32 AM said:
Thank you very much.