摘要
目的:建立和优化基于伤寒沙门菌全基因组DNA芯片的基因表达谱分析技术。方法:提取伤寒沙门菌野生z66阳性菌株总RNA,以6,9核苷酸随机引物(N6、N9)和(或)基因组特异引物(GDP)反转录合成cDNA,用荧光素Cy5或Cy3直接掺入标记后,与包括伤寒沙门菌4201个蛋白编码基因的伤寒沙门菌全基因组芯片杂交,用芯片扫描仪扫描后获得表达谱分析结果,经过数据标准化处理后进行分析。结果:采用N9+GDP混合引物,直接掺入法标记时,既能获得较好标记效果,又能节约试剂成本,同时可减少繁琐步骤所引起的不必要失误。结论:建立的基于伤寒沙门菌基因组DNA芯片的基因表达谱分析技术,为细菌的基因表达调控及功能基因组学研究提供了平台。
Objective： To develop and optimize the Salmonella enterica serovar Typhi genomic DNA mieroarray-based gene expression profiling. Methods： Total RNA of the z66-positive wild strains of Salmonella enterica serovar Thypi was extracted and reverse-transcribed to eDNA with random hexa-oligonucleotide primers （ N6 ） , nona-oligonucleotide primers （ N9 ） and genome-directed primers （GDP）. During reverse-transcription, eDNA was labeled with Cy3-dye or Cy5-dye directly. The labeled probes were purified and hybridized to a genomic DNA microarrays containing 4201 ORF of S. enterica serovar Typhi. The images were obtained by a laser scanner with two channels and the digital data were analyzed by the Acuity 4.0 software. Results： Using N9 ＋ GDP primers and direct labeling methods could not only obtain the excellent expression profiling results, but also eut down the cost of the reagent and avoid the errors caused by the multiple steps. Conclusion： The Salmonella enterica serovar Thypi genomic DNA microarray-based gene expression profiling was developed, which provides a platform for gene transcriptional regulation and functional genomics studies.
出处
《江苏大学学报（医学版）》
CAS
2009年第4期281-285,289,共6页
Journal of Jiangsu University：Medicine Edition
基金
江苏省高校自然科学基金指导项目(1191270005)
关键词
伤寒沙门菌
基因组
DNA芯片
基因表达谱
Salmonella enterica serovar Typhi
genomic DNA microarrays
gene expression profiling
