Unusual background problems on Southern blots

David Steffen steffen at hawk.bcm.tmc.edu
Fri Aug 10 09:44:50 EST 1990


  We have recently been having a problem with our Southern blots which in some 
ways acts like repetitive sequences in the affected probes, but in other ways 
doesn't.  I wonder if anyone else reading this group has experienced similar 
problems, and what they did about it.

  We have isolated a series of clones from a region of the rat genome (Dsi-1).  
All of these clones have been screened for rat repetitive sequences by 
hybridization of the clone DNAs on a filter to radiolabled rat DNA, and by this 
criterion have been found to be free of repeats.  When we then use the clones 
as probes to screen DNA from a variety of species (rat, mouse, hamster, rabbit, 
human) we find that some of the clones produce nice blots as expected, but that 
two of the probes produce a high background reminiscent of repeats.  What is 
odd is that the background is NOT seen in rat DNA, and is seen in only SOME of 
the other species.  My previous experience with repeats in probes is that 
background is only a problem with DNA of the same species as the probe was 
derived from (resulting from rapid evolution of repetitive sequences).  Even 
given an evolutionarily conserved repeat, why should it be present in only one 
copy in the species of isolation (rat) but in many, many copies (producing a 
smear on the blot) in some other species (mouse, hamster)?  Not suprisingly, we 
are considering the possibility that this is some kind of technical artifact, 
but we haven't been able to think of what kind.  Any suggestions along those 
lines would be welcomed as well.

  If you have any insight, please respond to me by email, and I will post a 
summary of any responses to this newsgroup.  Even if I receive no response, I 
will summarize that fact (after about two weeks).  Obviously, responses 
directly to this newsgroup are infinitely preferable to no response.

  Thanks in advance!

-David Steffen- (steffen at mbir.bcm.tmc.edu)



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