PCR technique for deletions-Questions

fsajb3 at aurora.alaska.edu fsajb3 at aurora.alaska.edu
Thu Jul 28 00:43:21 EST 1994


I am interested in trying out a PCR technique I heard about some time ago. 
I  want ot delete certain regions in our gene of interest and I I have heard of
a 4 primer PCR technique that sounds really nifty. Basicly two PCR reactions
are done that generate products on either side of the region you want to
delete. The inside primers are designed so that they have overlapping sequence.
These PCR reactions are then combined and amplified again with the outside
primer pair to give a final product with the deletion in the middle somewhere.
My questions are: How much overlap is required on the inside primers for this to
work? Is there a size limit where this technique is no longer viable (we want
to knock out ~100bp from a 3kb region)? If anyone has info as to publications,
or personal experience I'd love to hear about it.
 
Thanks,
Austin



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