ARS | Publication request: A NEW DNA OLIGO MICROARRAY FOR COMPLETE GENOME OF PSEUDOMONAS FLUORESCENS PF-5

Submitted to: International Union of Microbiological Societies Proceedings/Abstracts
Publication Type: Abstract
Publication Acceptance Date: July 28, 2005
Publication Date: July 23, 2005
Citation: Liu, Z., Duboy, R.T., Press, C., Loper, J.E., Paulsen, I.T., Slininger, P.J. 2005. A new DNA oligo microarray for complete genome of Pseudomonas fluorescens pf-5 [abstract]. International Union of Microbiological Societies. Abstract No. B-1258, p. 63-64.

Technical Abstract: Background: Pseudomonas fluorescens Pf-5 is a soilborne bacterium that functions as a biological control agent, colonizing root surfaces and protecting them from infection by plant pathogenic fungi. It produces a suite of antibiotics including pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol. Methods: DNA sequence of P. fluorescens Pf-5 genome was completely analyzed, and 7.1 Mb high-quality sequences were identified. A total of 6,147 unique DNA oligo 70-mer nucleotides representing the entire Pf-5 genome were designed and synthesized with an amino modifier at the 5¿ end. A set of six control genes were tested and selected. DNA oligo 70-mer nucleotides for each control gene were designed and synthesized with the amino modifier at the 5¿ end. The Pf-5 gene microarray was fabricated using GeneMachine OmniGrid 300 microarrayer robot. Results: Using a novel array design, we generated a DNA oligo microarray consisting of 19,000 elements representing 6,147 genes of the entire genome of P. fluorescens Pf-5. The Pf-5 gene chip had three replicated spots for each of the 6,147 genes located in separated blocks and contains our newly developed quality controls. We developed exogenous nucleic acid controls using ACTB, B2M, and PGK1 from Bos taurus, and CAB, MSG, and RBS1 from Glycine max. Multiple control gene spots were evenly printed and distributed on the entire microarray slide. In addition, a prescan mini array consisting of the six control genes and two additional negative controls was incorporated on top of the Pf-5 gene microarray as a reference to adjust PMT Gain prior to a full scanning of the entire array. A preliminary functional annotation was obtained for the Pf-5 gene microarray. Conclusions: Our newly developed P. fluorescens Pf-5 DNA oligo microarray provides a useful tool for functional genomic studies of the bacterium. Our newly developed exogenous nucleic acid controls are necessary quality controls to secure data quality and reproducibility of microarray experiments. These external RNA controls quantitatively spiked in labeling reactions can be used as a reference to perform normalization and data acquisition, evaluate array sensitivity, estimate hybridization consistency, and measure array quality and variance of microarray experiments.