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how to remove a restricted endonuclease site


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#1 WOW

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Posted 16 March 2009 - 06:21 PM

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.
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#2 perneseblue

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Posted 16 March 2009 - 06:30 PM

For PstI, a single bp change to its restriction site would be sufficient to knock out the site.
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#3 phage434

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Excellent

Posted 16 March 2009 - 06:32 PM

This strategy will work fine. But the DNA is not a protein(!).

#4 WOW

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Posted 16 March 2009 - 06:39 PM

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(!).


Thank you very much.
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