An oligonucleotide microarray was designed at the James Hutton Institute (formerly SCRI), and synthesized by Agilent Technologies, Inc. (Palo Alto, CA). Each slide contains 8 arrays and each array has nearly 15,000 spots, containing our probes in triplicate. The main E. amylovora ATCC 49946 genome (accession NC_013971, Sebaihia et al. 2010) had 3483 target sequences (annotated genes and pseudo-genes), plus a further 483 target genes or simple gene predictions from five sequenced plasmids: plasmids 1 and 2 for the same strain (accessions NC_013972, NC_013973, Sebaihia et al. 2010), pEL60 and pUE30 (accessions NC_005246, NC_005247, Foster et al., 2004), and pEI70 (Spanish strain Erwinia amylovora IVIA1614-2a, unpublished, Lopez and Llop, personal communication). Due to the high similarity between E. amylovora ATCC 49946 plasmid 1 (NC_013972, 2.8kb) and the previously published pEA29 (NC_005706, 2.8kb, McGhee and Jones 2000), the later was not used. Up to five sense orientation candidate probes per target were designed with the Agilent eArray webtool, using temperature matching methodology, a preferred probe melting temperature of 80.0?C, no 3' bias, and a target length of 60bp. Any short probes were later extended to 60bp using the Agilent linker. BLASTN (Altschul et al., 1997) and Biopython (Cock et al., 2009) were used to identify potential cross-hybridization in order to rank the candidate probes. Selecting one probe per genome target, and up to five probes per plasmid target, allowed all our probes to be present in triplicate. Using the spare array capacity for multiple plasmid probes aimed to increase the chance of hybridization with plasmid transcripts from other E. amylovora strains.
Description
An oligonucleotide microarray was designed at the James Hutton Institute (formerly SCRI), and synthesized by Agilent Technologies, Inc. (Palo Alto, CA). Each slide contains 8 arrays and each array has nearly 15,000 spots, containing our probes in triplicate. The main E. amylovora ATCC 49946 genome (accession NC_013971, Sebaihia et al. 2010) had 3483 target sequences (annotated genes and pseudo-genes), plus a further 483 target genes or simple gene predictions from five sequenced plasmids: plasmids 1 and 2 for the same strain (accessions NC_013972, NC_013973, Sebaihia et al. 2010), pEL60 and pUE30 (accessions NC_005246, NC_005247, Foster et al., 2004), and pEI70 (Spanish strain Erwinia amylovora IVIA1614-2a, unpublished, Lopez and Llop, personal communication). Due to the high similarity between E. amylovora ATCC 49946 plasmid 1 (NC_013972, 2.8kb) and the previously published pEA29 (NC_005706, 2.8kb, McGhee and Jones 2000), the later was not used. Up to five sense orientation candidate probes per target were designed with the Agilent eArray webtool, using temperature matching methodology, a preferred probe melting temperature of 80.0C, no 3' bias, and a target length of 60bp. Any short probes were later extended to 60bp using the Agilent linker. BLASTN (Altschul et al., 1997) and Biopython (Cock et al., 2009) were used to identify potential cross-hybridization in order to rank the candidate probes. Selecting one probe per genome target, and up to five probes per plasmid target, allowed all our probes to be present in triplicate. Using the spare array capacity for multiple plasmid probes aimed to increase the chance of hybridization with plasmid transcripts from other E. amylovora strains.
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