DNA Hybridization with complex probes

Dom Spinella dspinella at chugaibio.com
Wed Aug 27 10:44:40 EST 1997


Here's a question for any hybridization experts out there. Suppose one
wanted to perform a "reverse Northern" to confirm and quantify expresion
of a gene (or more likely a panel of dozens to hundreds of genes) in
mRNA derived from a particular cell or tissue.  If you have a panel of
cloned genes dotted onto a nitrocellulose filter grid and you prepare a
32P labelled total cDNA probe from the mRNA in question (say from 2 ug),
what would be the minimum copy number of the specific message in the
mRNA which would be required to give a detectable signal on the blots? 
Clearly there are C0t calculations that are relevant here and it would
depend on the volume of the hybridization fluid and the time as well as
the copy number.  I suppose this is why the "gene on a chip" approach,
in which hybridization can take place under a cover slip in a few ul,
seems to work for even low abundance messages.  However for the rest of
us who perform hybridizations in volumes of say 5-10 ml, can you detect
relatively low abundance messages (on the order of 10 copies per cell)
this way? Another way of phrasing this question is to think about a
screen of a complete cDNA library (say 150,000 lambda plaques lifted
onto a few filters) with the homologous total cDNA probe. Would every
clone in the library light up? Anybody have any practical experience or
tips (or maybe references)?  Thanks a lot.  -- D.G. Spinella



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