title: A CAPS-based binding assay provides semi-quantitative validation of protein-DNA interactions.
abstract: Investigation of protein-DNA interactions provides crucial information for understanding the mechanisms of gene regulation. Current methods for studying protein-DNA interactions, such as DNaseI footprinting or gel shift assays, involve labeling DNA with radioactive or fluorescent tags, making these methods costly, laborious, and potentially damaging to the environment. Here, we describe a novel cleaved amplified polymorphic sequence (CAPS)-based binding assay (CBA), which is a label-free method that can simplify the semi-quantitative validation of protein-DNA interactions. The CBA tests the interaction between a protein and its target DNA, based on the CAPS pattern produced due to differences in the accessibility of a restriction endonuclease site (intrinsic or artificial) in amplified DNA in the presence and absence of the protein of interest. Thus, the CBA can produce a semi-quantitative readout of the interaction strength based on the dose of the binding protein. We demonstrate the principle and feasibility of CBA using B3, MADS3 proteins and the corresponding RY or CArG-box containing DNAs.
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou 510642, China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, South China Agricultural University, Guangzhou 510642, China.|Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, South China Agricultural University, Guangzhou 510642, China.|College of Life Sciences, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Yongyao
authors.initials: Y
authors.lastname: Xie
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou 510642, China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, South China Agricultural University, Guangzhou 510642, China.|Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, South China Agricultural University, Guangzhou 510642, China.|College of Life Sciences, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Yaling
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou 510642, China.|Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, South China Agricultural University, Guangzhou 510642, China.|College of Life Sciences, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Xiucai
authors.initials: X
authors.lastname: Zhao
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou 510642, China.|Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, South China Agricultural University, Guangzhou 510642, China.|College of Life Sciences, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Yao-Guang
authors.initials: YG
authors.lastname: Liu
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou 510642, China.|Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, South China Agricultural University, Guangzhou 510642, China.|Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, South China Agricultural University, Guangzhou 510642, China.|College of Life Sciences, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Letian
authors.initials: L
authors.lastname: Chen
mesh_terms: D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011485:Protein Binding
chemical_list: D004268:DNA-Binding Proteins; D004247:DNA