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PCR technique for deletions-Questions

yd128 at albnyvms.bitnet yd128 at albnyvms.bitnet
Thu Jul 28 17:15:47 EST 1994


In article <1994Jul27.214321.1 at aurora.alaska.edu>, fsajb3 at aurora.alaska.edu writes:
>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
             Hi:  That method is basically a recombinent PCR.  The size of
deletion is limitless.  In the second round PCR, the middle overlap region
should be around 20 bp.  The critical part is to isolate PCR products from
template at each step.  Otherwise, you will get a mixture of deletion and
non-deletion PCR r products if your deletion region is quite small, which you
can not identify the difference between deletion and non-deletion in the
agarose gel.  Soory  for the misspeeling, this program can not edit the mail
once it is on the screen.  If you have further question, let me know.




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