help single copy gene detection with FISH

lorraine sohlberg lorraine at NATURE.BERKELEY.EDU
Wed Oct 9 14:17:34 EST 1996


Hello, I am interested in starting a project to localize a single 
copy gene on maize, corn, meiotic pachytene chromosomes.  We have 
localized a repetative sequence with FISH and now want to see if we 
can detect a single copy gene. I have heard that really large probes 
are needed and that the probe can not contain any repetative element 
in an intron or spacer region that will light up other sequences in 
the genome. I am also curious about the type of probe synthesized. I 
have seen people use nested sets of random oligo labelled probes 
thinking that the small probes will hyb first and that the longer 
probes would have a overhanging tail which then would hybridize to 
other labelled strands creating a tree of probe at the site. Sounds 
complicated. In situ PCR may be useful to generate signal too but I 
have no experience with this. We have been using DIG labelled RNA 
probes.  Thanks for the help in advance,  Lorraine



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