cloning biotinylated DNA's
jim at AUDREY.LMF.ORG
Mon Sep 27 19:10:11 EST 1993
I wish to suclone a 400 bp part of the human genome and analyse it for UV
induced mutations. Even with repeated exposure of cells to high doses, at
best 1 in 1000 of these fragments amplified by PCR would have a mutation.
So my problem is how to enrich the cloning process for mutation containg
fragments. On way I have considered is to use a biotinylated nucleotide
in the PCR reaction that corresponds to the expected C to T UV mutation.
Two reports in the litterature say that even if only one extra analog
is incorporated it can be separated by a change in electrophoretic mobility.
I could then just isolate all shifted bands and clone them into a vector.
The question I have is when I transfect E.coli with these biotin containing
clones will the grow in E.coli and will they be replicated accurately so
that mini-preps from isolated colonies will yield the original sequence??
Any experience or references on subcloning DNA's with multiple biotin analogues?
Is there a better way to enrich for mutation containing DNA's for sequence
Thanks in advance
The Lovelace Institutes
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