steffen@hawk.bcm.tmc.edu (David Steffen) (08/10/90)
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@mbir.bcm.tmc.edu)