Genomic measurement of DNA-binding protein expression

DNA 结合蛋白表达的基因组测量

• 批准号: 6648965
• 负责人: Stephen Patrick Walton
• 金额: $ 2.08万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2003-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6648965
• 关键词: DNA binding protein; Escherichia coli; SDS polyacrylamide gel electrophoresis; biotechnology; gene expression; genetic markers; genetic techniques; genome; high performance liquid chromatography; human genetic material tag; microarray technology; molecular shape; nucleic acid quantitation /detection; polymerase chain reaction; postdoctoral investigator; protein protein interaction; protein sequence; proteomics; technology /technique development; transcription factor; yeasts

DESCRIPTION (provided by applicant): Inappropriate/unregulated expression of DNA-binding proteins (DBPs), in particular transcription factors, has been implicated in many diseases, including cancer, AIDS, and diabetes. Current methods for measurement of DBP expression are inadequate for simultaneous measurement of all relevant proteins. The work proposed will provide a genomics-based, parallel method for the measurement of DBP expression using unique genetic tags termed molecular barcodes. Molecular barcodes, developed for the analysis of yeast growth rates, are 20 base DNA sequences designed to minimize cross-hybridization and secondary structure. The barcodes have been synthesized within "cassettes" with universal forward and reverse PCR primers, allowing single-tube amplification of thousands of cassettes. Using a barcode specific microarray, simultaneous detection of all barcodes within the mixture is possible. With each barcode linked to a particular species within a mixture, detection of the barcode confirms the presence of the encoded species (e.g., the presence of a single strain of yeast within the pool of all possible strains). For the proposed work, new cassettes will be designed that incorporate the recognition sequences of DBPs. Initial experiments on single proteins will validate the protocol for separation of bound and unbound cassettes followed by detection by quantitative PCR. Subsequent experiments using complex mixtures of DBPs and cell extracts will be analyzed using the barcode microarray. The flexibility of application of the barcode cassettes will be demonstrated by simultaneously measuring expression of E. coil, yeast, and human DBP expression on a single microarray. Finally, mutation analysis will be used to determine the most critical bases involved in the DNA:DBP interactions.

描述（由申请人提供）：DNA结合蛋白（DBP），特别是转录因子的不适当/不受调节的表达与许多疾病有关，包括癌症、艾滋病和糖尿病。目前测量 DBP 表达的方法不足以同时测量所有相关蛋白质。拟议的工作将提供一种基于基因组学的并行方法，使用称为分子条形码的独特遗传标签来测量 DBP 表达。分子条形码是为分析酵母生长速率而开发的，是 20 个碱基的 DNA 序列，旨在最大限度地减少交叉杂交和二级结构。条形码是在带有通用正向和反向 PCR 引物的“盒”内合成的，允许单管扩增数千个盒。使用条形码特异性微阵列，可以同时检测混合物中的所有条形码。每个条形码都与混合物中的特定物种相连，条形码的检测证实了所编码物种的存在（例如，在所有可能的菌株池中存在单一酵母菌株）。对于拟议的工作，将设计新的盒，其中包含 DBP 的识别序列。对单一蛋白质的初步实验将验证结合和未结合盒的分离方案，然后通过定量 PCR 进行检测。使用 DBP 和细胞提取物的复杂混合物进行的后续实验将使用条形码微阵列进行分析。条形码盒应用的灵活性将通过同时测量单个微阵列上大肠杆菌、酵母和人 DBP 表达的表达来证明。最后，突变分析将用于确定 DNA:DBP 相互作用中涉及的最关键碱基。