restriction enzymes for AFLP
Mr JN Goulding
jgouldin at hgmp.mrc.ac.uk
Thu Oct 5 03:55:19 EST 2000
On Fri, 29 Sep 2000, Thomas Moen wrote:
> I was thinking I should try some enzyme combinations other than EcoRI/MseI
> for the AFLP procedure:
> 1) Does anyone have any recommendations they would like to share with me?
> Or are there any enzymes I should avoid?
The adaptors are designed (assuming you are following the protocol in the
original Vos paper (I don't have the ref to hand I'm afraid)) so that they
make use of the 'HANDS' technology (again no ref to hand) which means
that eco-eco fragments and mse-mse fragments are not amplified- you only
get eco-mse fragments. If you are going to design new adaptors for
different restriction enzymes I would recommend basing the design on the
Eco/Mse adaptors and only change the restriction overhang section. This is
what I did for when I used alternative enzymes.
> > 2) Seems to me it would be a good idea to pick enzymes that are not
> methylation-sensitive. Then why is PstI one of the the most frequently used
> 6-cutters after EcoRI ?
I also used enzymes that were not methylation sensitive. Another thing to
bear in mind is enzymes that do not cut every restriction site but are
only cut depoending on the surroundiung seq. NarI is like that, and I
believe NaeI. Also try and pick enzymes whose restriciton site is
destroyed after the ligation step (Sau3A1 is one whose restriction site is
not destroyed). If you use these, remember to denature the enzymes before
the ligation step.
> 3) I've read somewhere that the C in CG is more prone to mutation (to T)
> than the average nucleotide. Is that so? What about C not in CG. Are there
> other short sequences that are mutated more often than expected?
> And if there are anyone out who'd like to discuss AFLP-related stuff, please
> don't hesitate to send an email!
Hope this helps, and if you want any more info then feel free to email me
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