SAP

PSB

Structure of the SAP promoter amplicons

Each promoter amplicon was defined in silico by a pair of oligonucleotides designed for PCR synthesis. The amplicon 3' end was delimited by the nucleotide preceeding the translation initiation codon in the corresponding gene model. Because multiple transcription initiation sites can be defined for individual genes, the amplified fragments also include the corresponding 5' untranslated region. The amplicon 5' end was located at the boundary between the targeted intergenic region and the adjacent upstream open reading frame (ORF), or ca. 2.0 kb upstream of the translation initiation codon.

General Overview

The amplicons were synthesized in a two-step PCR with genomic DNA (Columbia 0 ecotype) as template. The oligonucleotides used for amplification introduce a nucleotide triplet immediately upstream of the promoter specific sequences. The triplets can be used to differentiate PCR products in a multiwell plate. The amplicons are also flanked by attB4 (5') and attB1 (3') sites designed for site-specific recombinational cloning with additional DNA fragments via Multisite Gateway reactions. The initial cloning step to capture a SAP promoter into a Gateway entry clone is to recombine the amplicon with the pDONR P4-P1R plasmid in a BP reaction. For more information about the Multisite Gateway system consult Cheo et al., 2004 , Dupuy et al., 2004 , Karimi et al., 2005 , Karimi et al, 2007 and www.invitrogen.com

The promoter collection can therefore be used for a variety of application requiring cloning, such as yeast one-hybrid interaction study, promoter::reporter analysis, transactivation assay or ectopic expression. Because all promoter amplicons carry the same 5' and 3' extensions, the collection can also be reamplified straightforwardly and printed onto microarrays used in combination with chromatin immunoprecipitation for the analysis of chromatin-associated proteins or chromatin modifications.