I am trying to see the role of DENGUE NS1 trough PCR based mutagenesis approach by mutating some of the gene elements responsible for secretion of the protein into the blood stream. Is it possiible by mutagenesis? Please advise me.
In which form do you have the viral genome first of all. And yes if you know the active site then at the right spot even one point mutation can give non functional protein. But my best bet would be to introduce a stop codon somewhere in the ORF.
Hello Zeeshan,
Thanks for the answer. We made a dengue viral NS1 plasmid constructs of and try to do some mutational studies.
Yes then it's text book maneuver with the PCR mutagenesis. There are some commercial kits available for this. But all in all it is a straight forward procedure. You do the PCR, kill the template DNA with Dpn-I digestion, phosphorylate the ends and ligate it back.
Now where to mutate is tricky, if you dont know the structural biology and active site then I would suggest try to put a stop codon in the middle of the ORF.
But why are you not trying to look at the function by comparing a empty vector and vector with gene of interest at first to see if it works, in my opinion it is much easier. Or am I missing some experimental model details.
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