Cloning from genomic DNA - (Jun/28/2008 )
Does anyone use a reliable cloning protocol that allows isolation of a protein from genomic DNA (prokaryotic) by PCR if only the amino acid sequence is known (or at least point me in the right direction). The protein has three subunits, so it will probably require me to clone into a pET DUET vector or something.
You sure want to do this?
It may take a lifetime of effort developing a novel method, and even then you may not be able to do this particular thang.
Yes, I realize it will probably take some time. But it has to be done!
You need to be able to amplify a fragment of DNA from your selected gene. Once you have done this, you can expand the region easily with inverse PCR. The best approach is to locate highly conserved regions of your gene. Within those regions, locate amino acid residues with unique codons, such as met (atg) and trp (tgg). These unique codons serve as anchor point in constructing a degenerate primer pair which will prime to the conserved region. Amplifying with the degenerate primer, followed by sequencing will provide sufficient sequence for doing inverse PCR to expand the sequenced region to encompass the entire gene.
So these unique amino acid residues should be in the 3'-end of the degenerate primers, right?
I presume you've gone to http://www.genome.jp/kegg-bin/create_kegg_menu or similar to see if the genome has been done.
You can set up degenerate primers, using the unique codons mentioned by the phage-meister to anchor the sequence, and do a touchdown PCR to improve your chances of getting the best sequence. Use a proofreading polymerase to reduce errors further.
Yes, put the least degenerate codons preferably at the 3' end. AA's with two or four codons are not so bad -- Gly gives a ggn in the primer. Looking at codon usage for the organism can give you help in choosing the degenerate primer, since rare codons will be, well, rare. You probably want to avoid AA sequences with six-codon codes suh as Leu and Arg.
Thanks for the info guys! I'll give it a shot.