Quantifying DNA-protein binding specificities by using oligonucleotide mass tags and mass spectroscopy

@article{citeulike:1196763, abstract = {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. 10.1073/pnas.0611075104}, author = {Zhang, Lingang and Kasif, Simon and Cantor, And C. }, citeulike-article-id = {1196763}, doi = {http://dx.doi.org/10.1073/pnas.0611075104}, journal = {PNAS}, keywords = {dna, massspectroscopy, oligonucleotide, protein}, month = {February}, number = {9}, pages = {3061--3066}, posted-at = {2007-04-23 04:03:20}, priority = {2}, title = {Quantifying DNA-protein binding specificities by using oligonucleotide mass tags and mass spectroscopy}, url = {http://dx.doi.org/10.1073/pnas.0611075104}, volume = {104}, year = {2007} }

TY - JOUR ID - citeulike:1196763 L3 - citeulike-article-id:1196763 TI - Quantifying DNA-protein binding specificities by using oligonucleotide mass tags and mass spectroscopy JF - PNAS VL - 104 IS - 9 SP - 3061 EP - 3066 N2 - 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. 10.1073/pnas.0611075104 KW - dna KW - massspectroscopy KW - oligonucleotide KW - protein AU - Zhang, Lingang AU - Kasif, Simon AU - Cantor, And PY - 2007/02/27/ UR - http://dx.doi.org/10.1073/pnas.0611075104 ER -