PCR smear

Frederick Garbrecht FRED at bmt.mcw.edu
Mon Sep 26 08:26:50 EST 1994


I am attempting to map a potential mutation in a patient by using 
RT-PCR and a set of overlapping primers.  One of the forward primers, 
when used in combination with several of the other reverse primers 
(using the patient cDNA as template) always seems to give rise to a
smear on agarose gel electrophoresis, and no discrete bands at all
can be seen.  In contrast, the same conditions using control cDNA 
give rise to the expected size bands.  Is it reasonable to 
hypothesize that the sought after mutation may actually be present at 
the forward primer site, and that the smear occurs because the primer 
cannot bind there but does possibly misprime at lots of other places? 
Or is there some other obvious explanation that I am missing?  I 
think the patient template cDNA is reasonably OK, since I can amplify 
several other fragments that I have tried.

Frederick C. Garbrecht
fred at bmt.mcw.edu
Bone Marrow Transplant Program
Medical College of Wisconsin
phone 414 257 5053
fax   414 257 7994



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