AFLP, phosphorylation and restriction enzymes
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Fri Jul 16 03:37:34 EST 1999
In article <378E53B2.9E9A5BD7 at cdc.gov>, Scott Handley <ssh4 at cdc.gov>
>Most protocols do not call
>for the adaptor design to include a 5 prime phosphate additionally added
>to the oligo. I know that the ligation will still occur without a 5
>prime phosphate beign attached to the adaptor, and I can still get the
>AFLP to work. However, it is not ver robust, and I have been playing
>with other enzymes. My quesiton is: Do some restriction endonucleases
>affect the ability for an adaptor sequence to ligate? Do they leave the
>over-hang or blunt-end in such a way as to affect the ability for T4 DNA
>Ligase to do it's job?
To ligate two DNA fragments only one has to have a 5' phosphate. If you
kinase the adaptor it will ligate to itself etc. so don't do that!
Sticky overhangs ligate more efficiently than blunt, however enzymes
with single base overhangs appear to ligate very inefficiently. Have a
look at the NEB catalogue. It will give you an idea as to how efficient
the ligation is for each enzyme. I would definitely use enzymes with
I'm sure other people doing AFLP will be able to offer much more advice.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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