Have a mutant? Want the gene? AFLP clone it!
LISCUM at ANDREW.STANFORD.EDU
Mon Dec 11 21:56:11 EST 1995
As many of you probably know, a new DNA fingerprinting
technology has been developed by Marc Zabeau and
colleagues at KeyGene in the Netherlands (Zabeau and Vos,
1993, European Patent Appl, pub # EP 0534858-A1; Vos et al,
1995, NAR 23: 4407-441) that represents undoubtedly the
most powerful mapping technology available today. We
have been using this technology, known as AFLP (amplified
fragment length poylmorphism), for about a year now in or
efforts to clone genes identified by mutation that affect
phototropic responses of Arabidopsis. Several papers have
appeared recently that attest to the power of AFLP with
respect to generating markers tighly linked to loci of interest,
and doing so in short periods of time with alot less work
than required for RFLP mapping (Thomas et al., 1995,
Plant J 8: 785-794; Meksem et al., 1995, MGG 249: 74-81
Becker et al., 1995, MGG 249: 65-73).
It is estimated that AFLP has the potential to allow one to
visualize 3000-5000 polymorphic markers in Arabidopsis.
With a current map of around 500 RFLP markers, and the
time and energy required to RFLP map, it seems crazy not
to consider AFLP as an alternative to your upcoming
chromosome walk. With AFLP a walk really becomes a
As one example in our lab, we have used AFLP to go from
a F2 population segragating for one of our nph
(nonphototropic hypocotyl) loci to wild-type lambda clones
spanning the locus of interest in just around nine (post-doc)
months (once we worked some bugs out of the methods).
All of this occurred without markers linked to the genetic or
physical maps. We are convinced that AFLP represents the
future of mapping and positional cloning approaches, as it
is extremely robust and very rapid and reproducible, and
for these reasons represents the most cost-effective means
of getting to your gene of interest (that's the most important
bottom line right?).
Given all of the above soap boxing, I would like to inform
all those interested that we have placed our modified
AFLP protocol on the Carnegie-Dept of Plant Biology
WWW page. The protocol gives background information,
detailed AFLP protocol, and our "next steps" to the protocol
describing cloning of the linked DNA fragments and their
use for screening libraries.
I encourage people who are considering an RFLP-based
chromosome walk to their mutant gene, to take a look at our
web pages. Chances are pretty good that once you've done
that you will (or should) and then go back to the lab and start
running AFLP gels, and forget about that two, three,....year
chromosome walk. Under a year sounds much better doesn't
Cheers and Happy Cloning
PS. I plan to keep updating the AFLP page, so if people use
this technology and find new twists that make life easier or
more efficient, pleas let me know and I will add addendum
accordingly. I think it is important to keep this information
up to date and accessable to everyone.
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