restriction enzymes for AFLP

Mr JN Goulding jgouldin at hgmp.mrc.ac.uk
Thu Oct 5 04:05:08 EST 2000


Distrubution of fragment length depending uopon the enzymes used will
depend upon the organism studied, so therefore so will choice of
enzymes. I've had very good sucess with EcoR1/MseI and NarI/Sau3AI (though
these have problems of there own) and less joy with ApaI/TaqI. Others in
teh lab found ApaI/TaqIbest for their organism.

jon

On 4 Oct 2000, Mark Berres wrote:

> In article <8r248k$sub$1 at snipp.uninett.no>,
> 	"Thomas Moen" <thomas.moen at akvaforsk.nlh.no> writes:
> > Hi,
> > 
> > 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?
> 
> Hi Thomas,
> 
> My favorites are EcoRI + (ASE I or BFA I). These enzymes provide a much
> better distribution of fragments lengths, especially when compared to
> MseI. 
> 
> Although the subsection is not finished, I have an image comparing
> an EcoRI+MseI (on the left) and an EcoRI+AseI fAFLP (on the right; 
> the URL is given below).
> The pics should be under "Polymorphism screening with fAFLP".
> 
> > 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 ?
> 
> As far as I know, Pst I is not sensitive to classically understood
> methylation systems. However, I have noticed many researchers using
> TaqI (which is strongly sensitive to overlapping methylation). This
> is great if you are studying methylation patterns. I'm not quite
> sure what it means when screening for nucleotide polymorphisms...
> 
> As an aside, PstI,HindIII,MseI etc. were used in the original
> development of SRFA/AFLP. Also, ABI supplies their AFLP kit with
> EcoRI/MseI. I suspect that enzyme usage is simply a matter
> of historical and supplier convenience.
> 
> > 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?
> 
> A higher degree of C->T is expected as transitions >> transversions,
> typically ~x2. For CpG's, there is a deficiency (from expectation).
> This is due that CpG doublets are often methylated which elevates
> the mutation rate from C -> T (leading to higher than expected
> frequencies of TG and CA) due to spontaneous deamination. I'm not
> up-to-date on other remarkable doublet mutation biases other than
> the classic GC/AT pressures which many believe shape codon usage.
> 
> > And if there are anyone out who'd like to discuss AFLP-related stuff, please
> > don't hesitate to send an email!
> > 
> > Thanks,
> > 
> > Thomas Moen
> > AKVAFORSK - Institute of Aquaculture Research
> > Aas, Norway
> > thomas.moen at akvaforsk.nlh.no
> > 
> > 
> 
> -- 
> Mark E. Berres
> University of Wisconsin- Madison
> http://ravel.zoology.wisc.edu/sgaap
> 16 T 0303764
>      4771858
> 






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