Nonradioactive vs. radiolabelled probes

Wasun Chantratita asmsi002 at CMU.CHIANGMAI.AC.TH
Thu Mar 10 09:47:17 EST 1994


 
On Wed, 9 Mar 1994 jnppo at unity.ncsu.edu wrote:

> In article <Pine.3.87.9403032020.E25036-0100000 at cmu.chiangmai.ac.th> you wrote:
> 
> 
> : 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
> : ---------------------------------------------------------------------------
> 
> 
> Greetings,
> 
> I was wondering what size oligo do you use for the conjugation?  I would
> like to use (CAC)5 for fingerprinting.  Have you been able to use 15-mers? 
> 
> Thanks,
> 
> 
> James Petitte
> North Carolina State University
> J_Petitte at NCSU.eu
> 
Dear Jame
	I never try to couple alkaline phosphatase to oligonucleotide shorter 
than 20 mers. Is there any one out there has tried successfully to link 
alkaline phosphatase enzyme at 5' of 15 mers of oligonucleotide? I wonder 
whether it still function as a specific probe properly.


Wasun




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