Exo-Taq-Based Detection of DNA-Binding Protein for Homogeneous and Microarray Format

Abstract

The study of DNA-protein interactions is of great importance to understand basic cellular processes such as transcription, replication and recombination. In this research, we developed a novel detection system for DNA-binding proteins (DBPs) involving the exonuclease (Exo) III and Taq DNA polymerase reactions. The system consists of three steps, as follows: the target DBP in the sample solution is incubated with probe DNA, and the probe is digested with Exo III and then extended with Taq using fluorescent dye-labeled dUTP as a substrate. The DBP protects the probe from digestion by Exo III. Therefore, only the DBP-bound probe allows the following extension. We examined this system using the lambda phage Cro repressor in a homogeneous format. The fluorescence image after gel electrophoresis showed a specific band. We also found that this system could be applied to the rapid and efficient detection of DBPs in stem and loop ds-DNA array formats. These results suggest that our method is useful as a new tool for analyzing DNA-protein interactions.

Techniques for assaying the activity of transcription factor NF-κB

“…TransFactor NF-κB Kit (BD Biosciences) 、 NF-κB [65] , TransAM ® NF-κB Chemi Transcription Factor ELISA Kits(Active Motif) [66] 等试 剂盒。与比色分析相比较, 化学发光灵敏性高(每孔 可检测<10pg 的 NF-κB p50)、线性范围宽(0~1000pg NF-κB p50/孔, r 2 >0.99)但其化学发光信号会随时间 延长而衰减 [56] 。 此外, 最近有研究报道可将 dsDNA 包被的微孔 板 (Cayman Chemical) 与 近 红 外 荧 光 标 记 的 抗 体 (Li-COR biosciences)相结合, 用于 NF-κB 活性的检 测 [67] 1979 年, Hart 和 Greenwald [99] 第一次提出邻近闪 烁分析(Scintillation proximity assay, SPA), Udenfriend 等 [100] [27] 。近年来, 我们也以转录因子 NF-κB 为 研究对象, 进行了转录因子活性及转录因子与 DNA 相互作用的高通量、高灵敏性检测技术的研究, 发 展了数种转录因子, 特别是 NF-κB 活性检测的新技 术, 包括优化的化学放光 EMSA [41] 及近红外荧光 EMSA [42] 实验技术、基于 NOS 基团修饰微孔板的 DPI-ELISA 实验技术 [60,61] 、ExoⅢ-ELISA 技术 [68] 、 ExoⅢ-FRET 技术及限制性内切酶-RCA 技术 [94] 。这 些技术都被成功用于 NF-κB 等转录因子的活性检 测。我们首次提出的基于核酸外切酶 ExoⅢ的转录 因 子 检 测 技 术 (Exo Ⅲ-FRET), 促 进 了 该 领 域 其 他 ExoⅢ相关检测技术的发展, 如 ExoIII-ELISA 技术 [68] 、 CCP-FRET 技术 [86] 、ExoⅢ-Sybrgreen 染色法 [90] 、 ExoⅢ分子开关技术等 [91] 、ExoⅢ-DNA 聚合酶检测 法 [92] 、ExoⅢ-定量 PCR 检测法 [93] 等, 受到国际同行 的高度评价 [102] 技术 [79] 、基于链竞争的 FRET 技术 [83] 、三链核酸 FRET 技术 [84] 及转录因子信标技术(TF-MB) [87]…”

Profiling RNA Polymerase–Promoter Interaction by Using ssDNA–dsDNA Probe on a Surface Addressable Microarray

“…[9] However, most of these techniques are complex and manually demanding to perform and are limited to studying small numbers of DNA-protein interactions. Recently, the use of high-throughput microarrays has gained considerable interest particularly for analyzing binding characteristics of transcription factors [10][11][12][13][14][15] and specificities of restriction endonucleases. [16,17] Chromatin immunoprecipitation on chip microarrays (ChIP-chip) and DNA microarrays are more often used for target identification of DNA-binding proteins than protein microarrays.…”

“…[18,19] The principal drawbacks with these methods are the possible loss of binding affinity due to direct protein labeling and limited availability of detection antibodies targeted against DNA-binding proteins (DBP), particularly for mutant proteins. [15] In the ChIP-chip system, DNA fragments bound to proteins might not be sufficiently enriched, and substantial artifacts due to suboptimal experimental conditions can further limit this approach. [20] In addition, the occurrence of protein-DNA interaction at the solid-liquid interface might restrict the accessibility of proteins to the binding site owing to protein denaturation and steric hindrance of the immobilized double-stranded DNA (dsDNA) or proteins.…”