期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2007
卷号：104
期号：9
页码：3061-3066
DOI：10.1073/pnas.0611075104
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The ability to determine the relative binding affinity of different transcription-factors (TF) to their DNA binding sites is fundamentally important for a comprehensive understanding of gene regulation. Here we present a general approach for multiplex quantification of DNA-TF binding specificities in vitro using oligonucleotide mass tag (OMT) labeling and mass spectroscopic quantification. An OMT is a short nucleic acid sequence with a distinct mass that can be resolved by a mass spectrometer. Each putative binding sequence is labeled with a unique OMT, and PCR amplification of OMTs is performed after removing nonbound DNA. Subsequently, a primer extension reaction is carried out, and the extension products are quantified by MALDI-TOF mass spectroscopy. Using the TF NF-{kappa}B P50, we have quantified the binding specificities of up to 15 binding sequences in a single assay. The results from the multiplex assay are consistent with data from the traditional gel shift assay. The approach allows the competitive binding of multiple DNA sequences to the given protein in a homogeneous reaction. By using the commercially available homogeneous MassEXTEND platform (SEQUENOM), it is scalable for high-throughput DNA-TF binding applications, including genome-wide TF binding site mapping and analyses of SNPs in promoter regions.
