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CATMA, the Complete Arabidopsis Transcriptome Micro Array. Jim Beynon Horticulture Research International.

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Presentation on theme: "CATMA, the Complete Arabidopsis Transcriptome Micro Array. Jim Beynon Horticulture Research International."— Presentation transcript:

1 CATMA, the Complete Arabidopsis Transcriptome Micro Array. Jim Beynon Horticulture Research International

2 The Collaboration P. Hilson T. Altmann S. Aubourg J. Beynon - GARNet F. Bitton M. Caboche M. Crowe - GARNet P. Dehais H. Eickhoff E. Kuhn S. May - GARNet W. Nietfeld J. Paz-Ares W. Rensink P. Reymond P. Rouzé U. Schneider C. Serizet A. Tabrett - GARNet V. Thareau M. Trick - GARNet G. van den Ackerveken P. Van Hummelen P. Weisbeek M. Zabeau

3 GARNet GST Project Complement NASC EST arrays Add approx. 9000 GSTs Milestones:Start April 2000 2,500 GSTsApril 2001 6,000 GSTsApril 2002 9,000 GSTsMarch 2003 HRI:Jim Beynon, Alex Tabrett JIC:Martin Trick, Mark Crowe

4 AFGC Amplification project 3000 GSTs to be amplified HRI carried out 1000 amplifications Delivered to NASC April 2001 Remaining primers available at HRI but superseded by CATMA project

5 Goals of CATMA Produce Genome Sequence Tags (GSTs) for all predicted genes in Arabidopsis accession Columbia To use these GSTs to produce a complete genome Microarray.

6 EuGène genomic fragment genes RepeatMasker BlastnBlastx Netstart NetGene2 SplicePredictor Predicting the genes in Arabidopsis

7 EuGene Results on Chromosome V AGI: 5888EuGene: 6715

8 SPADS gene sequence exon coordinates GST Blastn Primer3 GST specificity primer specificity SPADS:Specific Primer and Amplicon Design Software

9 SPADS Results on Chromosome V AGI: 5888EuGene: 6715GSTs: 4784 (73%)

10 Distribution of GSTs lengths 150-200 bp: 42% 200-300 bp: 36% 300-500 bp: 22%

11 Position of GSTs ATGstop 5’ center3’ 3267 (16%) 5115 (24%) 12701 (60%) UTRCDS

12 Easy Re-amplification 16 x 24 U 3’ row 2 U 5’ col. 4 No cross contamination between wells

13 Genomic BAC DNA S 5 ’ U 5 ’ S 5 ’ U 3 ’ S 3 ’ First round PCR with specific primer pairs

14 U 3 ’ U 5 ’ Primary amplicon Second round PCR with universal primer pairs

15 Current Status of CATMA Genes identified within CATMA29,775 genes SPADS has designed21,420 GSTs GSTs as % of total genes72% Total Primary amplifications16,280 GSTs % Success in processed plates90.2% % Success after plate correction96%

16 CATMA: The Future SPADS has yet to design primers from 28% of genes identified within CATMA Extending 150bp into 3’UTR will probably yield a further 10% of GSTs Highly homologous genes will never yield GSTs Design best possible and note on website

17 CAGE: Compendium of Arabidopsis Gene Expression Having constructed the CATMA arrays we need to standardise them We are developing a project to produce a compendium dataset This will be available over the Web to enable users of CATMA arrays to relate their data to that of others Hughes et al. (2000) Cell 95, 717-728 Functional discovery via a compendium of expression profiles

18 RNAi: A key tool for future Arabidopsis research Insertional knockouts are limited in their use RNAi is a complementary aproach CATMA GSTs are ideal for RNAi We propose to produce a seed resource with RNAi constructs for all genes


20 probe length (bp) signal 1 10 100 1000 10000 100000 100600110016002100 FLOWER BUD Signal According to Length predicted gene GST known gene GST intergenic region GST highly expressed cDNA negative control

21 Success of EuGene actual genes correct gene models partial gene models split genes missing genes actual exons missing exons 238182 (76%) 50 (21%) 5 (2%) 1 (0.5%) 163951 (3%)

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