(based on protocols from Vos et al. 1995. Nuc. Acid Res. 23:4407-4414, Greg Penner, AAFC, Winnipeg and the
Digoxigenin system of Enrico Perotti, CIMMYT)
This AFLP protocol has been optimized for hexaploid wheat but has worked very well for maize, rye, tetraploid
durum wheat and Tripsacum. The use of PstI instead of EcoRI is especially useful for hexaploid wheat due to
the very large genome size and the very high level of repetitive sequences. PstI being methylation sensitive
results in fewer bands than an enzyme like EcoRI.
The chemiluminescent system described here consists of using one of the two selective primers labeled with
digoxigenin. After amplification and electrophoresis on sequencing gels, the amplification products are
transfered to a nylon membrane, and the anti-Dig / alkaline phosphatase and CSPD system is used to detect on
an X-ray film the amplification products.
The steps involved are:
– DNA digestion with two enzymes,
– Ligation of adaptors to restriction fragments,
– Pre-amplification using primers with one selective base for each restriction enzyme,
– Selective amplification using primers with three selective bases for each restriction enzyme, one of which is
– Electrophoresis in sequencing gels,
– Transfer of amplified fragments,
– Detection, exposure of membrane to X-ray film and development of X-ray film.
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