Arabidopsis EST Mapping Project
EXPRESSED SEQUENCE TAG (EST) ANCHORING OF THE ARABIDOPSIS
THALIANA YEAST ARTIFICIAL CHROMOSOMES (YACs).
Francis AGYARE1, Gus LAGOS1, Apostolos LAGOS1, Deval LASKARI2,
Ronald DAVIS2 and Bertrand LEMIEUX1
1Biology Department, York University, 4700 Keele Street, North York, Ontario
M3J 1P3 Canada.
2Center for DNA Sequencing and Technology, Stanford University, Palo Alto
California 94305 USA.
We are mapping Arabidopsis ESTs onto YAC clones using an entirely
PCR-based screening approach. The sequence data generated by the MSU and
French cDNA sequencing projects serves as our source of EST sequences.
Oligonucleotides for PCR amplifications are produced at Stanford University
using AMOS (Lashkari et al. 1995; Proc. Natl. Acad. Sci. 92:7912). Presently,
1,908 sets of primers have been synthesized and tested on Arabidopsis DNA and
65% of these EST primers give PCR products with plant DNA template. DNA
samples isolated from 4 Arabidopsis genomic YAC libraries (i.e., the CIC, yUP,
EG & EW available from the Arabidopsis Biological Stock Centers) have been
pooled according to the J-detector scheme (Balding 1994. Proc. Roy. Soc. Lond.
B 344: 329). Our screening is performed in two steps. In the first, we screen
primary pools consisting of 96 primary pools, with DNA from 36 clones in each,
which are organized into 3 sets of 10 J-detector pools. In the second, ESTs which
map to a given primary pool of 36 clones are used to screen the 10 J-detector
sub-pools derived from this primary pool. Both a positive and a negative control
consisting of plant DNA and yeast DNA templates, respectively, are included
with each experiment. Therefore the entire set of 3456 YAC clones can be
screened for one EST with no more than 72 PCR reactions. Considering that
each J-detector shares no more than three clones in common and that a given
clone is found in 5 pools, a YAC clone which contains an EST can be identified
with either 4 or 5 PCR products. Therefore, we have a built-in tolerance for one
false negative. The advantage of this method is that the redundancy of J-detector
pools compensates for the experimental noise commonly associated with PCR
based detection of sequences. We have written a computer algorithm which
allows the data to be compiled and processed in a semi-automated manner.
Currently, we have mapped over 271 ESTs on YACs using this approach.
This work was supported by grant GO-12403 from the Canadian Genome
Analysis and Technology (CGAT) program to B.L. and by grant NIH1P01
HG00205 from the National Institutes of Health (NIH) to R.W.D.
Arabidopsis EST Primer Plates
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NOTE: Program script for the automated assembly of the J-detector pools will be available shortly. Many thanks for your patience.
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Problems, suggestions or comments concerning our mapping project?
Send e-mail to Roger Lew (planters at yorku.ca)