Probing native DNA on filters(??)
Cheung C. Yue
ccy at po.CWRU.Edu
Fri Sep 18 20:25:08 EST 1992
In a previous article, bsh at MED.PITT.EDU (Basavaraju Shankarappa) says:
>> My question may be simple, but we don't seem to have
>> anyone here who has attempted this. Has anyone had any experience
>> with blotting native DNA (ie. Chromosome digest, non-denatured)
>> onto nylon filters and subsequently probing this DNA with a DNA
>> binding protein? I understand it is simple to blot native DNA
>> but will UV crosslinking fix it to the filter without having
>> previously denatured it?
>> One would assume that a DNA binding protein would require
>> the DNA binding site to be 1) in native form and 2) free from
>> binding to the filter. It would be nice if this method could
>> work and be used with a chromosomal digest blot or (even better)
>> a lambda bacterial library via colony hybridization.
>> G. Scott Gordon
>> Tufts U. - Sackler School
>Is this not the same as South-Western blot;
I don't believe what was asked was the South-Western blot. In that
one, the DNA-binding protein is fixed on the blot and probed with a
labelled DNA probe. Depending on what the original poster's intents
are, perhaps another useful technique is that of whole genome PCR
which I think was developed by Vogelstein's lab. In that technique,
DNA is fragmented, ligated to amplification primer-adapters, then
subjected to binding selection on immobilized DNA binding protein.
Bound DNA are rescued and amplified by PCR for additional rounds of
selection. Finally selected DNA fragments are cloned and sequenced.
Such fragments are obviously useful for screening genomic libraries.
ccy at po.cwru.edu
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