A-MEXP-1891 - R6-TIGR-Zerfass-alternative
The S. pneumoniae R6/TIGR4 -Chip The R6/TIGR4-Chip is a combined S. pneumoniae R6/TIGR4 Oligoarray-set, designed by the department of microbiology (University of Kaiserslautern) and produced in cooperation with the company Operon. Oligonucleotides are 70-mer sense oligos and have a 5’-amino-C6-linker. The oligoarray-chip consists of 2963 oligonucleotides in total (2347 oligonucleotides covering genes and unique intergenic regions which are longer than 200 bp of S. pneumoniae R6, 488 oligonucleotides covering genes and unique intergenic regions which are longer than 200 bp of TIGR4, 44 oligonucleotides covering additional sequences (repetitive elements, RNAs (tRNAs, rRNAs, RNaseP, small cytoplasmic RNA, small stable RNA) and one oligonucleotide for the pbp2x variant of the penicillin resistant isolate S. pneumoniae 2349, a representative of the clone Spain23F-1 (2349.2x)). 84 oligonucleotides represent controls: 12 randomized negative controls (HumV3con_1), 4 eucaryotic control genes, 10 positive controls and 4 stringency controls (4 oligonucleotides each with 100%-, 90%-, 80%- and 70%-match to the corresponding gene), 32 tracking oligos (opHsV04NC000001) representing randomly generated 30-mers (tracking oligos are randomly positioned in the 384-well (4 in each)) and 10 alien-controls according to the Stratagene SpotReport? Alien? Oligo Array Validation System (internal controls for the whole procedure (mRNA – cDNA- hybridisation). There are 4 types of oligonucleotides: type 1. Identical genes in R6 and TIGR4 (homology greater 95%); type 2. Genes occuring only in one of the two strains; type 3. Variable genes: one oligonucleotide covering the region conserved in both strains; type 4. Genes, which vary over the whole length (on DNA-level approx. 18-30 %): strain R6 is reference. Altogether 2038 oligonucleotides represent R6/TIGR4 genes and 309 oligonucleotides are TIGR4 specific; 328 oligonucleotides represent intergenic regions of R6 and 160 those of TIGR4. For some experiments an additional 384-well was used, containing 90 oligonucleotides (oligonucleotides for the six S. pneumoniae PBP-genes, the small RNAs 1-5, controlled by the two-component system CiaRH, rpsM, spr1094, ruvI 1-4, aorfR and stringency controls) as well as 32 buffer controls. Each oligonucleotide was spotted in duplicate onto Schott Nexterion? slide E using the SpotArray TM24 Microarray Spotting System BioChip Technologies (Packard BioScience) and the respective software SpotArrayTM (PerkinElmer).
Martin Rieger (firstname.lastname@example.org)