Genomic measurement of DNA-binding protein expression

描述（由申请人提供）：dna结合蛋白（DBPs），特别是转录因子的不适当/不调节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。提出的工作将提供一种基于基因组学的平行方法，使用称为分子条形码的独特遗传标签来测量DBP的表达。分子条形码是为酵母生长速率分析而开发的，包含20个碱基DNA序列，旨在最大限度地减少交叉杂交和二级结构。条形码在“卡带”中合成，使用通用的正向和反向PCR引物，允许单管扩增数千个卡带。使用条形码专用微阵列，可以同时检测混合物中的所有条形码。每个条形码与混合物中的特定物种相关联，条形码的检测确认了编码物种的存在（例如，在所有可能的菌株池中存在单个酵母菌株）。对于所建议的工作，将设计包含dbp识别序列的新卡带。对单个蛋白质的初步实验将验证结合和未结合磁带分离的方案，然后进行定量PCR检测。随后的实验使用DBPs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。条形码卡带应用的灵活性将通过在单个微阵列上同时测量E. coil，酵母和人DBP表达来证明。最后，突变分析将用于确定DNA:DBP相互作用中最关键的碱基。

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.