GLOBAL COMPETITIVE PCR FOR STUDYING GENE EXPRESSION

描述：(申请人的描述)这项工作的长期目标 研究是研究全球范围内的变化，即全基因组范围的基因表达 人体组织中与癌症、衰老和暴露相关的模式 对环境的压力。 在人体组织中研究的具体情况是，他们需要 处理大量样本和提取信息的能力 从少量的起始材料。现有的衡量方法 基因表达要么集中在少数几个靶基因上，要么 相对费力，或/和需要相当数量的样品。 迫切需要一种能够扩增整个mrna的程序。 从较小的起始量开始，并将与 DNA杂交等劳动效率高但耗费mRNA的方法 数组。问题是，在放大复杂混合物的过程中 诸如信使核糖核酸池的序列，单个序列的丰度 可能存在不可预测的偏见。 我们建议开发一种“全球竞争性聚合酶链式反应”程序，该程序将 扩增从待比较样本中提取的整个mRNA库 结合在一个反应管中。该方法基于 附加样品特定的标签序列和一对共同的 对待扩增池中的每个mRNA序列进行引物扩增。如中所示 竞争性聚合酶链式反应，相同(除标签外)模板之间的比率 来自不同的样本，因此关于 相对基因表达水平，在扩增过程中保持不变。 经扩增后，来自不同样品的序列 根据它们所承载的标签和 杂交到DNA阵列，以测量每个基因的比例 两个样本中的表达水平。这些比率可以进一步 转换成绝对的表达水平。 一组细菌DNA模型系统的初步实验 不同长度的序列验证了密钥的可行性 拟议程序的步骤。

DESCRIPTION: (Applicant's Description) The long term objective of this research is to study changes in global, i.e. genome-wide gene expression patterns in human tissues associated with cancer, aging, and exposure to environmental stresses. The specifics of studies in human tissues are that they require the ability to process a large number of samples and to extract information from small amounts of starting material. Existing approaches to measure gene expressions either concentrate on a few target genes or are relatively laborious, or/and require substantial amount of sample. There is an urgent need for a procedure that would amplify entire mRNA pools from small starting amounts and would be compatible with such labor-efficient but mRNA-consuming approaches as hybridization to DNA arrays. The problem is that during amplification of a complex mixture of sequences such as an mRNA pool the abundances of individual sequences may be unpredictably biased. We propose to develop a "global competitive PCR" procedure that will amplify the entire mRNA pool extracted from the samples to be compared combined in a single reaction tube. The approach is based on the attachment of a sample-specific tagging sequence and a pair of common primers to each mRNA sequence in the pools to be amplified. As in competitive PCR, ratios between identical (except for the tag) templates originating from different samples and thus the information about relative gene expression levels, is preserved during amplification. After amplification, the sequences originating from different samples are differentially labeled according to the tags they bear and are hybridized to a DNA array to measure, for each gene, the ratio of its expression levels in the two samples. The ratios can further be converted into absolute levels of expression. Initial experiments on a model system involving a set of bacterial DNA sequences of different length demonstrated the feasibility of the key steps of the proposed procedure.