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）的不适当/不调节表达，特别是转录因子，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。提出的工作将提供一种基于基因组学的平行方法，用于使用称为分子条形码的独特遗传标签来测量DBP表达。开发用于分析酵母生长速率的分子条形码是20种基本DNA序列，旨在最大程度地减少杂交和二级结构。条形码已在“盒式”中与通用前向PCR引物合成，从而可以单管扩增数千盒。使用条形码特定的微阵列，可以同时检测混合物中所有条形码。当每个条形码与混合物中的特定物种相关联时，条形码的检测证实了编码物种的存在（例如，在所有可能菌株的池中存在单个酵母菌菌株）。对于拟议的工作，将设计新的盒式盒子，以结合DBP的识别序列。对单蛋白的初始实验将验证该方案以分离结合和未结合的盒，然后通过定量PCR检测。随后的实验使用DBP和细胞提取物的复杂混合物将使用条形码微阵列进行分析。通过同时测量单个微阵列上的E. coil，酵母和人DBP表达的表达，可以证明使用条形码盒的灵活性。最后，将使用突变分析来确定DNA：DBP相互作用中涉及的最关键碱基。