Design optimisation and validation of high density microarrays for multiple Escherichia coli genomes

多个大肠杆菌基因组高密度微阵列的设计优化和验证

• 批准号: BB/F00396X/1
• 负责人: Charles Penn
• 金额: $ 8.49万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF00396X%2F1
• 关键词: Design; optimisation; validation; density; microarrays

Since the first complete DNA (genome) sequence for a free-living organism was determined for the bacterium Haemophilus influenzae in 1995, a wealth of additional genomes have now been analysed. They include about 40 from different Escherichia coli strains, creating the widest ranging genomic data set available for any species. These data now enable dissection, at the level of expression and its control, for every one of the 5000 or so genes found in E. coli. Genome sequences are available for strains ranging from important foodborne pathogens to vehicles for expression of medically important proteins for treatment of disease. New methods have been developed for exploitation and analysis of sequence data, with the generic name 'functional genomics'. This means analyses that are based on 'massively parallel' experimental designs where expression of each individual gene in the genome is analysed simultaneously using an array of specific detection probes, one for each gene, set out on a solid surface. Until recently, this approach was available for studies of transcription (formation of messenger RNA prior to production of the protein encoded by that gene) but had not been developed further. Now, colleagues in this School have worked out ways to use probe arrays to detect the locations of specific proteins called transcription factors. These factors bind to the chromosome to regulate the transcription of specific genes by facilitating or obstructing the enzymes involved. This new technology depends critically on technical advances in array fabrication, and our collaborators at Oxford Gene Technology (OGT) are leaders in this field. Arrays used previously are made by 'spotting' a solution of DNA in the form of a short stretch of known sequence onto a glass slide, one spot for each gene to act as a probe for the complementary sequence present in mRNA. Now, methods have been developed by OGT whereby these probes are synthesized on the slide in situ, using inkjet printer technology to deliver tiny spots of chemical reagent to each 'feature' or area of the surface that will contain a given probe, to determine the unique order of nucleotides forming that probe. The new method allows for dramatically greater numbers of probes and reduced costs per probe compared with the spotting of pre-synthesised solutions. This means there are enough probes to cover the whole genome, and locations of bound transcription factors can be identified clearly by using the array to detect fragments of chromosome bound to the protein when it is recovered and purified from bacteria. To exploit the availability of numerous new E. coli genome sequences and the technical ability to analyse their bound transcription factors at high resolution, we propose to design and validate, in collaboration with OGT, a new generation of high resolution microarrays. These will be optimized for both gene transcription studies and transcription factor binding (ChIP-on-chip) studies. The latter method is named after the first step to purify transcription factor protein bound to DNA: the protein DNA complex is called chromatin (abbreviated Ch); IP represents ImmunoPrecipitation, a method incorporating a specific antibody to recover just the one protein of interest from whole cell homogenates. 'Chip' represents the microarray (sometimes referred to as a 'chip' in analogy to IT terminology) used to analyse the DNA fragments bound to the immunoprecipitated protein. The work we propose will make this extremely powerful technical approach widely available to the research community and will underpin important advances in understanding of the ways E. coli controls expression of its genes, whether acting as a pathogen or being exploited in industrial processes. An exciting spin-off from the work will be unique and informative data comparing the patterns of gene expression and RNA polymerase binding throughout the genomes of 8 different strains of E. coli.

自从1995年确定了流感嗜血杆菌的第一个完整的DNA（基因组）序列以来，现在已经分析了大量的其他基因组。它们包括来自不同大肠杆菌菌株的约40种，为任何物种创建了范围最广的基因组数据集。这些数据现在使得我们能够在表达和控制水平上对大肠杆菌中发现的5000个左右基因中的每一个进行解剖。杆菌基因组序列可用于从重要的食源性病原体到表达用于治疗疾病的医学重要蛋白质的载体的菌株。开发和分析序列数据的新方法已经发展起来，通称为“功能基因组学”。这意味着基于“大规模平行”实验设计的分析，其中使用一系列特异性检测探针同时分析基因组中每个基因的表达，每个基因一个，在固体表面上设置。直到最近，这种方法才可用于转录研究（在该基因编码的蛋白质产生之前形成信使RNA），但尚未得到进一步开发。现在，这个学院的同事们已经找到了使用探针阵列来检测被称为转录因子的特定蛋白质的位置的方法。这些因子与染色体结合，通过促进或阻碍相关酶来调节特定基因的转录。这项新技术在很大程度上依赖于阵列制造的技术进步，我们在牛津基因技术（OGT）的合作者是这一领域的领导者。以前使用的阵列是通过将一小段已知序列的DNA溶液“点”在载玻片上来制作的，每个基因一个点作为mRNA中存在的互补序列的探针。现在，OGT已经开发出了在载玻片上原位合成这些探针的方法，使用喷墨打印机技术将化学试剂的微小斑点传递到包含给定探针的表面的每个“特征”或区域，以确定形成该探针的核苷酸的独特顺序。与预合成溶液的点样相比，新方法允许显着增加探针数量并降低每个探针的成本。这意味着有足够的探针覆盖整个基因组，并且当从细菌中回收和纯化蛋白质时，可以通过使用阵列检测与蛋白质结合的染色体片段来清楚地识别结合的转录因子的位置。利用大量新的E.大肠杆菌基因组序列和技术能力，以高分辨率分析其结合的转录因子，我们建议设计和验证，与OGT合作，新一代的高分辨率微阵列。这些将被优化用于基因转录研究和转录因子结合（ChIP芯片）研究。后一种方法以纯化与DNA结合的转录因子蛋白质的第一步命名：蛋白质DNA复合物称为染色质（缩写为Ch）; IP代表免疫沉淀，一种掺入特异性抗体的方法，从全细胞匀浆中回收一种感兴趣的蛋白质。“芯片”代表用于分析与免疫沉淀蛋白结合的DNA片段的微阵列（有时与IT术语类似称为“芯片”）。我们提出的工作将使这种极其强大的技术方法广泛应用于研究界，并将支持对E.大肠杆菌控制其基因的表达，无论是作为病原体还是在工业过程中被利用。这项工作的一个令人兴奋的副产品将是独特的和信息丰富的数据，比较了8种不同的大肠杆菌菌株基因组中基因表达和RNA聚合酶结合的模式。杆菌