A modified sensor chip for surface plasmon resonance enables a rapid determination of sequence specificity of DNA‐binding proteins

FEBS Letters

Ohme-Takagi, Masaru¹  Yamasaki, Kazuhiko¹  Hao, Dongyun¹ 

[1] National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan

关键词: Random oligonucleotide selection;    Surface plasmon resonance;    Sensor chip;    GCC-box;    DBD;    DNA-binding domain;    dsDNA;    double-stranded DNA;    EMSA;    electrophoresis mobility shift assay;    ERF;    ethylene-responsive element-binding factor;    FITC;    fluorescein isothiocyanate;    NTA;    nitrilotriacetic acid;    PAGE;    polyacrylamide gel electrophoresis;    PCR;    polymerase chain reaction;    SPR;    surface plasmon resonance;    ssDNA;    single-stranded DNA;   

DOI  :  10.1016/S0014-5793(03)00045-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A novel method is described which rapidly determines specificity of DNA-binding proteins using a surface plasmon resonance (SPR) sensor chip. An oligohistidine-tagged DNA-binding domain of a transcription factor, NtERF2, was immobilised via nitrilotriacetic acid ligands to a sensor chip with an attenuated degree of carboxymethylation. DNA molecules were selected from a pool of randomised oligomers through binding to the immobilised protein and amplified by PCR. After several cycles of selection, during which binding was monitored by SPR, DNA sequences containing a consensus sequence were determined. The time necessary for one cycle is ∼50 min, which is shorter than existing methods.

【 授权许可】

Unknown