restriction enzymes for AFLP

Mark Berres markb at ravel.zoology.wisc.edu
Tue Oct 3 22:56:46 EST 2000


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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