Need help amplifying repeating sequence.pcr repeat-rich sequence
Posted 24 August 2011 - 01:16 PM
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!
Posted 24 August 2011 - 10:26 PM
Posted 25 August 2011 - 05:37 AM
Posted 25 August 2011 - 06:23 AM
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?
Posted 25 August 2011 - 07:20 AM
Or, design primers with restriction sites, phosphorylate primers to allow for annealing and put directly into expression plasmid. What do you think?
Edited by jennifer6271, 25 August 2011 - 07:35 AM.
Posted 25 August 2011 - 11:04 AM
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.