Nonradioactive vs. radiolabelled probes

Wasun Chantratita asmsi002 at CMU.CHIANGMAI.AC.TH
Thu Mar 3 09:18:24 EST 1994



On Thu, 3 Mar 1994, M K Bennett wrote:

> Marion Freistadt (mfreis at lsumc.edu) wrote:
> : Has anyone compared the sensitivity of non vs. radiolabelled probes in
> : southern blots, all else being equal?  We are having difficulty detecting a
> : single copy sequence used the Genius kit; but we know the gene is there by
> : PCR.  

Dear Marion

	You can hardly detect any single copy sequence used the Genius 
kit as the company claim. At lease in our hand it never work.


:I was thinking maybe I should go back to 32P.  Comments?

 There is no need for you to go back to 32P. Try to use alkaline 
phosphatase labeled (5')oligonucleotide probe. You can choose either 
Lightsmit(Promega) or ACE System (BRL). It is very sensitive. 
We can easily detect a single copy of gene from Sounther transfer.Right 
now we routinely use the sytem to detect various type of viruses from 
different clinical specimens.

:My limited experiance with non-radio probes says that they are always
less sensative than 32P.  I have never got them to work effectivly when
probing northerns, Southerns or plaque lifts (lots of targets).  In the
end the fact that generally you get your data with 32P leads me to always
> use it.  It would be nice however not to have to use it.

People always think that 32P probe are more sensitive than non-radio
probes. Yes, it is true if we are talking about non radioactive labeled
polynucleotide probe. However,it is incorrect to say that non-radioactive
labeled (direct coupling of enzyme to) oligonucleotide probe is less
sensitive than 32P Oligonucleotide probe. Usually, we may labled them
(polynucleotide probe)with either biotin or digoxigenin. Streptavidin
-alkaline phosphatase conjugate/antidigoxigenin-alkaline phosphatase
conjugate can create hybridization background. Thus you can not allow
colorimetric detection or chemiluminescent detection process keep
continuing beyond 2 hr. In case of enzyme (alkaline phosphatase) labled at
5' of oligoprobe, it is sensitive as 32P oligoprobe since there is no
hybridization background (from Sterptavidin -alkaline phosphatase
conjugate and antidigoxigenin-alkaline phosphatase conjugate). You can
have either colorimetric detection or chemiluminescent process develop
overnight if you want to.
	According to polynucleotide probe, as far as I know we could not 
label them directly with alkaline phosphatase since usually the 
polynucleotide probe have to be denatured (heated up at 100oC) before use 
and formamide in hybridization buffer could inhibit the enzyme activity.

Hope this will help
----------------------------------------------------------------------------
Wasun Chantratita
Department of Clinical Microbiology
Faculty of Associated Medical Sciences
Chiangmai University
Thailand
Email:asmsi002 at cmu.chiangmai.ac.th
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