Cloning the coding region of a protein
Posted 29 November 2010 - 01:34 PM
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 -
Posted 29 November 2010 - 03:04 PM
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.
Posted 30 November 2010 - 12:23 PM
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?
Posted 30 November 2010 - 02:34 PM