cloning biotinylated DNA's

Jim Gale 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
analysis????

Thanks in advance
Jim Gale
The Lovelace Institutes
Albuquerque NM




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