Design of whole-genome tiling microarrays for staphylococcus aureus NCTC 8325 and pseudomonas aeruginosa PA01

• Main Author: Tan, Steffe Jie Ing.
• Other Authors: Chang Wook, Matthew
• Format: Final Year Project
• Language: English
• Published: 2009
• Subjects: DRNTU::Engineering::Chemical engineering::Biotechnology
• Online Access: http://hdl.handle.net/10356/16371
• Institution: Nanyang Technological University

Optimal design of microarray probes has a direct impact on the reliability of microarray results. Since whole genome tiling arrays have vast potential in the biotechnology industry, it is desirable to design reliable tiling probes with good genome coverage. The objective of this project is to design whole-genome tiling microarrays for Staphylococcus aureus NCTC 8325 and Pseudomonas aeruginosa PA01. This report describes an approach to designing whole genome microarrays using the program eArray created by Agilent Technologies, including a discussion of the design process and criteria, as well as the creation and selection of probes. To obtain information about how to go about designing microarrays, literature review on microarray technology was conducted. From the literature review, much knowledge about the parameters that affected the quality of the microarray data was gained, as well knowledge of several generic approaches to designing microarrays. Gene expression probes and expression microarrays were created for both micro-organisms using Expression application in eArray to better understand the design process steps. Using the simple tiling application in the eArray program, 60-mer oligonucleotide probes were designed. Basic Local Alignment Search Tool (BLAST) application in another program ‘CLC Main Workbench 5.0’ was used to optimise the selection of probes. The results were analyzed to study the probes for homology and elimination of duplicate probes to prevent cross-hybridization and improve target specificity. BLAST analysis yielded a final total of 55,249 probes for P. aeruginosa PA01 and 23,188 probes for S. aureus NCTC 8325. These probes were then used to complete the design of whole genome tiling arrays for both micro-organisms in eArray for chromatin-immunoprecipitation and comparative genome hybridization applications. The probe design procedure described combines optimal thermodynamic properties with high target specificity. Future recommendations include tighter tiling to improve genome coverage, and refining probe selection criteria after experimental validation to improve the quality of probes and reliability of microarrays. This generic tiling microarray design procedure using eArray has the potential of improving genome coverage even in regions that are conserved in the bacteria genome.