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

自从1995年为流感嗜血杆菌确定了第一个自由生命有机体的完整DNA(基因组)序列以来，现在已经分析了大量额外的基因组。它们包括来自不同大肠杆菌菌株的约40个，创建了可用于任何物种的最广泛的基因组数据集。这些数据现在可以在表达和控制的水平上对在大肠杆菌中发现的大约5000个基因中的每一个进行剖析。基因组序列可用于从重要的食源性病原体到表达治疗疾病的医学上重要蛋白质的载体等各种菌株。开发了开发和分析序列数据的新方法，其通称为“功能基因组学”。这意味着分析是基于“大规模平行”的实验设计，在这种设计中，使用一系列特定的检测探针同时分析基因组中每个单独基因的表达，每个基因一个探针放在固体表面上。直到最近，这种方法还可用于转录研究(在产生该基因编码的蛋白质之前形成信使RNA)，但没有得到进一步的发展。现在，该学院的同事们已经想出了使用探针阵列来检测称为转录因子的特定蛋白质的位置的方法。这些因子结合到染色体上，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技术的进步，我们在牛津基因技术公司(OGT)的合作伙伴是这一领域的领先者。以前使用的阵列是通过将DNA溶液以一小段已知序列的形式‘点’到玻璃片上，每个基因一个点作为存在于mRNA中的互补序列的探针。现在，OGT已经开发出了一些方法，通过这种方法，这些探针是在载玻片上原位合成的，使用喷墨打印机技术将微小的化学试剂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，新方法允许显著增加探头数量并降低每个探头的成本。这意味着有足够的探针覆盖整个基因组，当蛋白质从细菌中回收和纯化时，通过使用阵列检测与蛋白质结合的染色体片段，可以清楚地识别结合转录因子的位置。为了利用大量新的大肠杆菌基因组序列的可用性和高分辨率分析其结合转录因子的技术能力，我们建议与OGT合作设计和验证新一代高分辨率微阵列。这些将被优化用于基因转录研究和转录因子结合(芯片上芯片)研究。后一种方法是以纯化与DNA结合的转录因子蛋白质的第一步命名的：蛋白质DNA复合体被称为染色质(简写为CH)；IP代表免疫沉淀，一种结合了特定抗体的方法，从全细胞匀浆中仅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质结合的DNA片段的微阵列(有时被称为“芯片”，类似于IT术语)。我们提出的工作将使这一极其强大的技术方法广泛适用于研究界，并将为了解大肠杆菌控制其基因表达的方式取得重要进展，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个不同菌株的基因组中基因表达和RNA聚合酶结合的模式。

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.