Reverse dot-blot

[Mr. P.F. Ravetto]

Hello,

I am currently developing a high resolution HLA-DRB1 molecular typing
system based on PCR amplication of the relevant polymorphic region of the
gene followed by dot-blotting of the products onto membrane and
hybridisation to panels of sequence specific oligo probes. In order to
ensure the specificity of the probes, DNA from cell lines of known HLA
types (XII HLA workshop) are used as controls in hybridisation. My problem
is that because of the level of polymorphism in HLA-DRB1 I am unable to 
get such positive control material to test all of the probes that I am
proposing to use. I was therefore thinking about the possibility of using
antisense (reverse) oligos as controls for hybrisation where no cell line 
DNA is available. This would involve, in theory, putting an poly-T tail
onto the reverse oligos and binding them to the membrane thus provided the
correct control sequence available for hybridisation. Can anyone tell me
a) whether this is feasable, b) how long should the tail be on my oligos,
and c) the best way of binding the tailed oligos to the nylon membrane.


Many thanks


Paul  

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Paul Ravetto. 
Department of Medical Genetics,
Immunogenetics Lab.,
University of Manchester,
St. Mary's Hospital Manchester.
0161-276-6469.

Email pravetto at hgmp.mrc.ac.uk
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