[Q] HELP ON CHOICE OF NON-RAD LABELLING/DETECTION SYSTEMS

Abreu Grobois Federico Alberto-ICML abreu at REDVAX1.DGSCA.UNAM.MX
Thu Nov 10 18:59:54 EST 1994


We are initiating a study of the genetic structure of marine turtle
populations along the Pacific coast of Mexico which will be carried
out upon the analysis of PCR amplified microsatellites and
sequences of mitDNA d-loops. The work will be important for the
establishment of proper management strategies.

Because of the inherent dangers and complications involved in the
storage and treatment of radioactive materials (which are non-
existent where we work) we have decided to utilize non-radioactive
systems for the detection of the results. Not having colleagues
near by to advise us, we are finding it difficult to decide on what
is the best labelling system for our needs and would greatly
appreciate any help people participating in the net could give us.

The primers we will be using have already been defined by
colleagues working with sea turtles elsewhere, although they are
being applied with radioactive markers. Nonetheless, it seems that
we can still adapt non-radioactive labelling/detection by 
  a.    incorporating a non-radioactively labeled primer by:
     i.      ordering a primer synthesized with the non-rad label (or
             reporter molecule) at the 5' end, or
     ii.     label the available primers oneself the 5' end with a
             commercial labelling kit
  b.    incorporate non-radioactively labeled NTP's into DNA via PCR

then there are, of course, various non-radioactive labeling/
chemiluminescence systems (ie. DIG/chemiluminescence,
biotin/chemiluminescence) and the alternative Promega silver stain.

I wonder if you could advice on the best alternatives, that is:
   1.     use of non-rad labelling+chemiluminescence or silver stain?
   2.     and, if non-rad labelling+chemiluminescence is thought the
          best alternative, which is the recommended method for
          incorporating the label into the PCR products?

  a.    in terms of costs
  b.    in terms of intensity/sensitivity of the assay
  c.    and does the answer differ if the purpose is assayal of
     i.      length polymorphism in microsatellite regions amplified
             by PCR, or
     ii.     sequencing of PCR amplified sections

Finally, what are the best companies (cheapest) to deal with?

Really appreciatefully yours,


Alberto Abreu
***********************************************************************
Dr. F. Alberto Abreu Grobois
Banco de Informacion sobre Tortugas Marinas (BITMAR)
Estacion Mazatlan
Instituto de Ciencias del Mar y Limnologia 
UNAM                               tel.    52 (69) 85-28-45,-46,-47,-48
Apartado Postal 811                fax.    52 (69) 82-61-33
Mazatlan, Sinaloa 82000            e-mail  abreu at redvax1.dgsca.unam.mx
MEXICO                             priv.   52 (69) 81-22-94
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