Cloning the coding region of a protein - (Nov/29/2010 )
Im a novice at bioinformatics and need a bit of help.
Ive been given a region of a protein sequence and been asked to clone the coding region for the protein. Ive now gotten the whole protein, its name, structure and the mRNA sequence coding for the protein. Im thinkin to use RT PCR to clone the protein. But how do I go about designing primers for this? Ive gotten a region of 5 amino acid in the MIDDLE of the protein (least number of codons) that Im using to design primers, so that gives me a forward and reverse primer. Do I need to take 2 regions - one in the begining and one at the end of the protein to design primers? Or will one region suffice?
- Mon -
If you have the mRNA sequence for the protein, look for the start site (ATG) and the stop site (TAA, TGA) and then design a 5' primer that starts at the ATG, and a 3' primer that stops at the stop codon (don't forget to reverse complement). These should amplify the full coding sequence for the protein, provided it isn't too big (Taq doesn't work well over about 3 kbp, but there are other polymerases that will such as pfu, KOD, Pwo).
If you are cloning for expression of the protein from the plasmid you need to have a kozak sequence before the start codon... this sequence helps the ribosome recognise the start codon and is usually the sequence GCCACCATG (where the ATG is the start codon for your protein).
You are also likely to be cloning by restriction enzyme (RE) digest, which means that you need to know what RE sites there are on the plasmid multiple cloning site and whether any of these are present in your coding sequence (in which case you can't use them). You can add the RE sites onto the 5' end of each primer, and if you use RE's that generate overhanging ends you can do directional cloning, meaning that the insert should always be in the correct orientation. If you are lucky or in a rich lab you may be able to do TA cloning which is really easy and quick - all you need to do is amplify the coding region, add an A overhang and ligate.
That was a great help!
But one confusion remains. I have the POSSIBLE coding region of the mRNA, which means for each amino acid I have worked out the possible number of codons and so the region that I will take in the begining (and at the end) will have many codons. Threonine for example will have ACT/ACC/ACG and so how do I work out the possible number of primers and also can I instead just take the least degenrate region somewhere in the middle of the sequence instead?
Ok, yes, you need to do some degenerate PCR initially to work out the coding sequence fully. I suggest doing 5' and 3' RACE (rapid amplification of cDNA ends) to work out the sequence. The techniques are a little tricky, but there are quite a few good kits out there (try invitrogen and/or Roche)
Thanks !! Your an angel