Separate DNA fragments of same length

Brian J. Baumgartner brianb at bcm.tmc.edu
Fri Feb 5 20:25:41 EST 1993


In article <1ksj7nINN5r6 at access.usask.ca>, lur at jester.usask.ca (Rui Lu) writes:
> Hi,
> 
>       Is there anyone knowing how to separate DNA fragments of exactly same
> length (about 150 bp) with different sequence? These fragments were isolated
> by PCR using degeneracy primers. I don't want to screen hundreds of colonies
> by DNA sequencing. Also, I don't want to lose many amplfication products.
> 
>       Any smart ideas?
> -- 
> Ray Lu
> lur at sask.usask.ca
> Ph:(306)966-4440/4442


You did not mention the extent of degeneracy of your primers, however I
have played around with hybridization of PCR products generated with
degenerate primers.  I found that oligos differing by as few as 4
nucleotides can be selective.  The stringency conditions have to be
carefully adjusted.  I have even heard of people obtaining selectivity
with only one nucleotide difference.  You will need a collection of the
various oligos with only one of the possible degenerate nucleotides at the
variant positions (in other words not a mixed population of degenerate
primers).  The Molecular Cloning Manual by Sambrook, et al has a good
discussion on hybridization with oligonucleotide probes.  This approach
would at least give you some information on which primers were used to
generate the PCR products.  You could also use this approach to screen
recombinants for different PCR products without having to sequence the
plasmids.  Techniques are available to PCR from single bacterial
colonies.  This would of course require you to clone the PCR products. 
See the many discussions on direct ligation of PCR fragments posted to
this newsgroup.

Good Luck
Brian
brianb at mbcr.bcm.tmc.edu



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