Summary: Non-radioactive detection of DNA

David Nunn David_Nunn at qms1.life.uiuc.edu
Sun Dec 13 12:01:45 EST 1992


A summary of answers to my recent post (below) in case any one is
interested, thanks to all who wrote. 

>   I'm sure that this same thread has been on the net before but forgive
>me. 
>   We are thinking of switching to non-radioactive Southern hybridization.I
>would like some feedback on which systems you have found to be the easiest
>to use, most cost effective, etc..  We would be using these methods
>primarily for clone screening and prokaryotic chromosomal DNA hyb's, so
>extreme sensitivity is not required.  If I get a few responses I will
>repost them to the net.  Thanks

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We have tried Amersham's ECL 3'-oligo labeling kit as an alternative to 
radiation and it has worked very the couple of times we've used it.  It was

one of the few "kits" that managed to work the first time when used by the 
book.   We weren't looking for anything rare, our Southern was an oligo 
that hybridized to digested fragments of a cosmid.  Unlike radiation, one 
can't follow the probe so until you go through the developing steps you 
don't know if you've inadvertantly washed your probe off - 

Soon, when some $$$ come in, we'll be trying the ECL random prime kit for 
Southerns and Northerns.  I don't know about anyone else's institution, but

here at Wash-U we're looking at about $500 everytime a 5 gallon drum of 
liquid radiation waste leaves the lab...  if for nothing else, that is 
gentle "push" to consider non-radioactive alternatives...

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I could recommend you Digoxigenin (Genius) system, which is ideal for
Southern
blotting with "random-prime" labelled DNA probe and hybridization with 7%
SDS.
I know people, which are very satisfied.
Please note, that DIG system is low efficient at Northern blotting, but at
Southern the efficiacy is like by 32p or better.

We switched to the Boehringer DIG kit some years ago, as I had a lab full
of people who really didn't want to work with radioactivity (young, of
female persuasion, child-bearing age, etc).  We have had nothing but good
experiences with it.  The kit seems expensive at first sight, but our cost-
and-buy lady worked it out as being cheaper than using (say) the
equivalent 32P kit, because (main reasons):

1) The probes, once labelled, last for EVER;

2) No radiaactives disposal or exposure problems.

Additionally, you can label up PCR products to high activity simply by
adding DIG-11-dUTP-containing nucleotide mix either from the kit, or from
a mix you make up yourself (from Promega nucs. and B-M DIG-dUTP) (for Jim
Graham, who doesn't like kits 8-)  ), and PCRing away normally.  Try that
with radioactivity - then explain to everyone who uses the machine after
you that no, it isn't hot...!

The detection kits (or make 'em up yourself, Jim) use either NBT-BCIP (or
other) insoluble dye product, OR use the proprietary (or make it up
yourself, Jim!!) TROPIX or Boehringer AMPPD chemiluminescence kit (see also
recent post on DIG-Northerns), which work VERY well, and give detection
close to the 32P limit and much quicker, in my experience (hours instead
of days).  You can also clean off probes, re-probe, re-expose, etc.

And no, I don't work for Boehringer, I just shamelessly solicit free
samples from them....

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The USB system works well.  Key is probe labeling.  Sequenase
synthesizes biotinylated probes which contain a higher proportion
of biotin than Klenow-made probes.

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David Nunn, Ph.D.
Department of Microbiology
University of Illinois
Urbana, IL 61801
(217) 333-6131



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