AFLP marker

[Gary C. Donaldson]

In article <moellere.12.00074D61 at pz350.ipsp.uni-hohenheim.de>, 
moellere at pz350.ipsp.uni-hohenheim.de
 says...
>
>Hello all,
>anyone on the net already has practical experiences with the AFLP marker 
>technique using the original KEYGENE protcol and/or the brandnew AFLP kit from 
>GIBCO/LIFE TECHNOLOGIES ?
>I want to use the technique for genotyping of  exotic plant species (not the 
>usual cash crops the kit is designed for) and for filamentous fungi.
>

Hi Evelyn,
I been AFLPing for about a year now.  I've used the technique a little with conifers, but 
largely with filamentous fungi, mainly Fusarium, but also Botrytis, Neurospora, 
Trichoderma, Sclerotinia, and a few others.  I've just followed the protocols outlined in 
the original patent application.  The enzyme/primer combinations I've used easily 
differentiate species of Fusarium, and I've found the technique to be very useful.  I 
originally began using these because I hate RAPDs....  too touchy and difficult to 
reproduce.  AFLPs have two key advantages over RAPDs:  first, you can vary the 
concentration of template DNA without seeing artefactual changes in banding pattern; 
and second, the use of the adapter makes the reactions less prone to amplify 
contaminating DNA (assuming you're cautious to avoid contamination when you do the 
restriction digest and adapter ligation step).  As with RAPDs, you do see some 
artefactual variation in banding patterns with changes in magnesium, primer, and dNTP 
concentrations, but certainly nowhere near the degree that you see in RAPDs.  Some 
variation in banding patterns also occurs using enzyme/buffer solutions from different 
vendors.  All of the artefactual effects are greatly reduced by hot-starting the reaction.  If 
you want reproducible results, hot-starting is a must.  I hope this helps.  If you have any 
more questions, drop me an e-mail.


----------------------------------------------------------------------------------
Gary C. Donaldson
Dept. of Plant Science, University of B.C.
E-mail:  garyd at unixg.ubc.ca
              donaldson at bcr.bc.ca
              72700.2227 at compuserve.com
-----------------------------------------------------------------------------------
There are three kinds of lies. 
Lies, damn lies, and statistics.               -Mark Twain
-----------------------------------------------------------------------------------