Tandem repeat deletion - an idea for preventing it (Mar/13/2008 )

Hey guys,

I have a gene with four x 48 bp tandem repeats in it and it keeps getting deleted when i clone it. I've tried using strains like SURE, SURE2, Stbl4 and growing at 30C but nothing has prevented the deletion. I was wondering what exactly causes the tandem repeats to become deleted. Recombination or looping out? How exactly does it work? From what i've read on the web they're not completely sure how it works either but i was hoping someone could shed more light on it.

The reason i ask is that i have an idea to prevent the deletion by altering the sequence of my gene (using the degeneracy of the genetic code) without changing its amino acid sequence. My only problem is that because im not quite sure how the deletion is occurring im not quite sure which sequence/s i should target. Would the outside repeats be better to target than the internal ones?

Any comments appreciated.

Cheers, Rob

SorryI don't know the answer, but maybe I came across something similar. I'm curious, what is the percentage of deleted clones? 100%? In my case some clones were deleted but some were intact. This was ok becasues I didn't plan any further manipulation of clones, just needed their sequence.

Sounds to me like the repeat region is forming a loop which is then not getting replicated, check for self complementarity of the repeat sequence and probably a little bit to either side of the repeats. You could try cloning each half of the gene separately into plasmids that can recombine if bought into proximity, otherwise no particular suggestions.

Thanks for the replies. Turns out i was using the wrong clone - a deleted clone - as my template when i amplified the gene to clone it into a new vector. Couldn't believe it, silly mistake but mistakes happen i guess. So it was 100% of the clones getting deleted. In response to you Bob, i did look at self complementarity of different parts of the repeat. Different parts formed stronger hairpin than others - the strongest areas were in the middle and on the ends of the repeats. For future reference, how would you clone the insert in two halves as you've suggested? Can you target the recombination without altering the sequence of the gene? I'm not sure it would work but am interested any way.