DOI: 10.3724/SP.J.1206.2008.00373

Progress in Biochemistry and Biophysics (生物化学与生物物理进展) 2009/36:2 PP.206-212

Preparation of Salmonella enterica Serovar Typhi Genomic DNA Microarrays for Gene Expression Profiling Analysis*

Salmonella enterica serovar Typhi, a gram-negative human enteroinvasive pathogen is the etiological agent of typhoid fever, and is an important experimental model for prokaryote research. DNA microarray technology was widely used in analysis of genomic structures and expression profiles. The genomic DNA microarrays were prepared based on the genomic sequences of S. enterica serovar Typhi. A total number of 4 201 protein encoding genes selected from strains Ty2, LT18 and a z66+ wild strain of S. enterica serovar Typhi were amplified by PCR. The products were purified and printed onto the poly-L-lysine coated chip slides in duplicate to form the genomic DNA microarrays. Fluorescently labeled probes were prepared by priming of genomic DNAs with random hexamers and extension with Klenow DNA polymerase. Labeled DNAs were hybridized with the microarrays to check the printing effect and verify the genes order. The genomic DNA microarray-based bacterial gene expression profiling analysis was optimized and used to investigate global transcriptional responses when wild strain of S. enterica serovar Typhi was grown in high- and low-osmotic environmental conditions. The major results were consistent with those of previous research with oligo-genomic microarrays of S. enterica serovar Typhi. These results demonstrated that S. enterica serovar Typhi genomic DNA microarrays were successfully prepared and could be utilized in relative gene expression profiling analysis and comparative genomic researches.

Key words:Salmonella enterica serovar Typhi, genomic DNA microarrays, gene expression profile

ReleaseDate:2014-07-21 14:48:40

Funds:This work was supported by grants from The National Natural Science Foundation of China (3057008) and The National Key Technology R D Program (2006BAK02A15).

1 Deng W, Liou S R, Plunkett G III, et al. Salmonella enterica subsp. enterica serovar Typhi Ty2 Complete genome information: http://www. ncbi. nlm. nih. gov/entrez/query. fcgi?db=genomeprj&cmd=Retrieve&dopt=Overview&list_uids=371, 2002-09-25

2 Everest P, Wain J, Roberts M, et al. The molecular mechanisms of severe typhoid fever. Trends Microbiol, 2001, 9(7): 316~320

3 Parry C M, Hien T T, Dougan G, et al. Review Article: Typhoid fever. N Engl J Med, 2002, 347(22): 1770~1782

4 Huang D V, DuPont H L. Problem pathogens: extra-intestinal complications of Salmonella enterica serotype Typhi infection. Lancet Infect Dis, 2005, 5(6): 341~348

5 Threlfall E J. Antimicrobial drug resistance in Salmonella: problems and perspectives in food- and water-borne infections. FEMS Microbiol Rev, 2002, 26(2): 141~148

6 Fodor S P A, Read J L, Pirrung M C, et al. Light-directed, spatially addressable parallel chemical synthesis. Science, 1991, 251(4995): 767~773

7 Agostini C, Miorin M, Semenzato G. Gene expression profile analysis by DNA microarrays: a new approach to assess functional genomics in diseases. Sarcoidosis Vasc Diffuse Lung Dis, 2002, 19(1): 5~9

8 Kim M J, Lim S, Ryu S, et al. Molecular analysis of the Salmonella Typhimurium tdc operon regulation. J Microbiol Biotechnol, 2008, 18(6): 1024~1032

9 Hinchliffe S J, Howard S L, Huang Y H, et al. The importance of the Rcs phosphorelay in the survival and pathogenesis of the enteropathogenic Yersiniae. Microbiology, 2008, 154(4): 1117~1131

10 Blixt O, Hoffmann J, Svenson S, et al. Pathogen specific carbohydrate antigen microarrays: a chip for detection of Salmonella O-antigen specific antibodies. Glycoconj J, 2008, 25(1): 27~36

11 Batchelor M, Hopkins K L, Liebana E, et al. Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria. Int J Antimicrob Agents, 2008, 31(5): 440~451

12 Chan K, Baker S, Charles C. Kim, et al. Genomic comparison of Salmonella enterica serovars and Salmonella bongori by use of an S. enterica serovar Typhimurium DNA microarray. J Bacteriology, 2003, 185(2): 553~563

13 Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual. 3rd. New York: Cold Spring Harbor Laboratory Press, 2001.6.4~6.11

14 Huang X, Xu H, Sun X, et al. Genomic oligo microarray analysis of global transcriptional expression by Salmonella enterica serovar Typhi during hyperosmotic stress. Int J Mol Sci, 2007, 8(2): 116~135

15 Talat A M, Hunter P, Johnston S A. Genome-directed primers for selective labeling of bacterial transcripts for DNA microarray analysis. Nat Biotechnol, 2000, 18(6): 679~682

16 周冬生, 杨瑞馥, 宋亚军, 等. DNA 芯片技术在微生物研究中的应用. 军事医学科学院院刊, 2002, 26(4): 294~300 Zhou D S, Yang R F, Song Y J, et al. Bull Acad Milit Med Sci, 2002, 26(4): 294~300

17 庞 昕, 韩延平, 周冬生, 等. 基于DNA 芯片的细菌基因表达谱技术的建立与评价. 中华微生物学和免疫学杂志, 2006, 26(2): 179~184 Pang X, Han Y P, Zhou D S, et al. Chin J Microbiol and Immunol, 2006, 26(2): 179~184

18 Vrana K E, Freeman W M, Aschner M. Use of microarray technologies in toxicology research. Neurotoxicology, 2003, 24(3): 321~332

19 周冬生, 韩延平, 戴二黑, 等. 鼠疫耶尔森菌全基因组DNA芯片的研制及用于比较基因组学分析. 解放军医学杂志, 2004, 29(3): 200~203 Zhou D S, Han Y P, Dai E H, et al. Med J Chin Peop Liberat Army, 2004, 29(3): 200~203