Need help amplifying repeating sequence. - (Aug/24/2011 )
I am in need of some help amplifying a region of my gene-of-interest. The sequence is comprised of approximately 75% repeating sequence and I want to amplify just a portion of it in order to generate a recombinant peptide. As expected, I get quite a smear when I use standard pcr conditions. I have tried increasing the temp as well as gel purifying the area of the approximate size I want and using that as a a template with no luck. One suggestion I received was to amplify the entire sequence (which I am able to do because the 5' and 3' ends are not composed of repeating sequences) and the treat with DNase I to chew away some of the sequence. Any suggestions or thoughts are greatly appreciated!
What type of repeat is the sequence and does it have multiple copies in the genome? If it has many copies in the genome, probably you have to design primers on its flanking non-repeat sequences and then TA clone the product. After obtaining the plasmid, try amplify the desired region. this should not give you smear.
You say that you want to amplify a region to generate a recombinant peptide. How long a peptide is that? If it is not too long you might be better off by assembling this region from primers rather than amplifying it from a template.
Thanks pcrman and prodes for your responses!
pcrman- The repeating sequence is specific for my gene and I have TA cloned the full-length form. That is actually what I have been using for my template and I still get a smear. The problem is that the repeating region is roughly 1200bp and I don't want my peptide that large. I want it around 120bp or so.
prodes - I want a 40 residue peptide, give or take a few. How might I proceed to assemble this from primers rather than template?
Another thought, could I use two 120bp primers containing an SP6 promoter sequence that are complementary to each other for an in vitro transcription reaction the follow with an in vitro translation reaction?
Or, design primers with restriction sites, phosphorylate primers to allow for annealing and put directly into expression plasmid. What do you think?
In your situation I will certainly try the assembly approach. The size is way smaller than what I assembled recently. You need to design overlapping, complementary primers and do a pcr with gradually increasing annealing temperature starting from maybe around 50degC up to probably about 65degC for about 30 cycles (you will need proofreading polymerase like Pfu and equimolar ratio of your primers).
On your second though, yes in principle you can do in vitro expression with a linear dna that has a promoter, shine-dalgarno, rbs, start codon, protein of interest, stop codon and terminator (basically any plasmid's expression cassette). You have to check how cost-efficient that would be doing by assembly vs cloning it into a vector and then using the vector for your assay. My feeling is that it would be better to assemble just your target sequence then clone it in a vector for 1) you can easily sequence an individual clone, 2) you can safely amplify it just by doing more minipreps rather than PCR that could introduce mistakes.