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Plant Physiol, February 2001, Vol. 125, pp. 513-518
Efficient Screening of Arabidopsis T-DNA Insertion Lines Using
Degenerate Primers1
Jeffery C.
Young,
Patrick J.
Krysan, and
Michael R.
Sussman*
Biology Department, Western Washington University, Bellingham,
Washington 98225 (J.C.Y.); and Madison Biotechnology Center, University
of Wisconsin, 425 Henry Mall, Madison, Wisconsin 53706 (P.J.K.,
M.R.S.)
The sequencing of the Arabidopsis plant genome is providing a
fuller understanding of the number and types of plant genes. However,
in most cases we do not know which genes are responsible for specific
metabolic and signal transduction pathways. Analysis of gene function
is also often confounded by the presence of multiple isoforms of the
gene of interest. Recent advances in PCR-based reverse genetic
techniques have allowed the search for plants carrying T-DNA insertions
in any gene of interest. Here we report preliminary screening results
from an ordered population of nearly 60,470 independently derived T-DNA
lines. Degenerate PCR primers were used on large DNA pools
(n = 2,025 T-DNA lines) to screen for more than one
gene family member at a time. Methods are presented that facilitated
the identification and isolation of isoform-specific mutants in almost
all members of the Arabidopsis H+-proton ATPase gene
family. Multiple mutant alleles were found for several isoforms.
1
This work was supported by the U.S. Department
of Energy (grant no. DE-F602-88ER13938) and the National Science
Foundation (grant no. DBI 9872638).
*
Corresponding author; e-mail msussman{at}facstaff.wisc.edu; fax
608-262-6748.
© 2001 American Society of Plant Physiologists
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