GLOBAL COMPETITIVE PCR FOR STUDYING GENE EXPRESSION

用于研究基因表达的全球竞争性 PCR

• 批准号: 6040679
• 负责人: Konstantin Khrapko
• 金额: $ 9.99万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6040679
• 关键词: clinical research; complementary DNA; gene expression; genetic techniques; human genetic material tag; human subject; messenger RNA; polymerase chain reaction; technology /technique development

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.

描述：（申请人的描述）本发明的长期目标是： 研究是研究全球范围内的变化，即基因组范围内的基因表达 与癌症、衰老和暴露相关的人体组织模式 to environmental环境stresses应力. 人体组织研究的特点是， 处理大量样本和提取信息的能力 从少量的起始材料。 现有的衡量办法 基因表达要么集中在少数靶基因上，要么 相对费力或/和需要大量样品。 目前迫切需要一种方法来扩增整个mRNA 从小的起始金额，并将兼容这样的 劳动效率高但消耗mRNA的方法，如与DNA杂交 阵 问题是在复杂混合物的放大过程中 序列的丰度，例如mRNA库， 可能会有不可预测的偏差。 我们建议开发一种“全球竞争性PCR”程序， 扩增从待比较样品中提取的整个mRNA库 组合在单个反应管中。 该方法是基于 样品特异性标签序列和一对共同的 引物与待扩增池中的每个mRNA序列相匹配。 如在 竞争性PCR，相同（标签除外）模板之间的比率 来源于不同的样本，因此， 相对基因表达水平在扩增过程中得以保留。 扩增后，来自不同样品的序列 根据它们所携带的标签被不同地标记， 与DNA阵列杂交，以测量每个基因的 两个样品中的表达水平。 所述比率可以进一步为 转化为表达的绝对水平。 涉及一组细菌DNA的模型系统的初步实验 不同长度的序列证明了密钥的可行性 拟议程序的步骤。