Su Xiaodi, Robelek Rudolf, Wu Yingju, Wang Guangyu, Knoll Wolfgang
Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602.
Anal Chem. 2004 Jan 15;76(2):489-94. doi: 10.1021/ac035175g.
MutS protein is a mismatch binding protein that recognizes mispaired and unpaired base(s) in DNA. In this study, we incorporate the MutS protein-based mutation recognition into quartz crystal microbalance (QCM) measurements for DNA single-base substitution mutation and 1-4 base(s) insertion (or deletion) mutation detection. The method involves the immobilization of single-stranded probe DNA on a QCM surface, the hybridization of target DNA to form homoduplex or heteroduplex DNA, and finally the application of MutS protein for the mutation recognition. By measuring the MutS binding signal, DNA containing a T:G mismatch or unpaired base(s) is(are) discriminated against perfectly matched DNA at target concentrations ranging from 1nM to 5 microM. Furthermore, the QCM damping behavior upon MutS-DNA complex formation is studied using a Network Analyzer. The measured motional resistance changes per coupled MutS unit mass (deltaR/deltaf) are found to be indicative of the viscoelastic or structural properties of the bound protein, corresponding to different binding mechanisms. In addition, the deltaR/deltaf values vary remarkably when the MutS protein binds at different distances away from the QCM surface. Thus, these values can be used as a "fingerprint" for MutS mismatch recognition and also used to quantitatively locate the mutation site.
MutS蛋白是一种错配结合蛋白，可识别DNA中错配和未配对的碱基。在本研究中，我们将基于MutS蛋白的突变识别方法纳入石英晶体微天平（QCM）测量，用于检测DNA单碱基取代突变和1至4个碱基的插入（或缺失）突变。该方法包括将单链探针DNA固定在QCM表面，使目标DNA杂交形成同源双链或异源双链DNA，最后应用MutS蛋白进行突变识别。通过测量MutS结合信号，在1 nM至5 μM的目标浓度范围内，含有T:G错配或未配对碱基的DNA与完全匹配的DNA能够被完美区分。此外，使用网络分析仪研究了MutS-DNA复合物形成时QCM的阻尼行为。发现每个结合的MutS单位质量的测量运动阻力变化（deltaR/deltaf）可指示结合蛋白的粘弹性或结构特性，这与不同的结合机制相对应。此外，当MutS蛋白在距QCM表面不同距离处结合时，deltaR/deltaf值会有显著变化。因此，这些值可作为MutS错配识别的“指纹”，也可用于定量定位突变位点。
