Lucarelli Fausto, Capponcelli Silvia, Marrazza Giovanna, Sangiorgi Luca, Mascini Marco
Department of Chemistry, University of Florence, via della Lastruccia 3, 50019 Sesto F.no, Florence, Italy.
Analyst. 2009 Jan;134(1):52-9. doi: 10.1039/b806514d. Epub 2008 Oct 18.
This paper describes the development of a highly selective single-nucleotide polymorphisms (SNPs) typing method based on the use of split hybridisation probes and demonstrates the concept through the electrochemical analysis of single-base mutations in actual patient samples. The requirement that two probes hybridised adjacent to one another to allow for stabilisation (via base-stacking) and binding of the allele-specific oligonucleotide (ASO), imparted highly stringent selectivity criteria to the assay. Simple rules for tuning the characteristics of such stacking/ASO probe pairs and achieve full mismatch discrimination at ambient conditions (with no need to strictly control the temperature) are provided. All genotyping experiments were indeed performed at room temperature, using the planar surface of disposable probe-modified gold electrodes as the genosensing platform. The ability to detect nanomolar amounts of a synthetic target even within a vast excess of single-base substituted sequences gave strong evidence of the specificity of the split probes assay. Proving the general validity of this genotyping approach, application of the analytical pathway was further demonstrated for clinical targets (amplified from the human TP53 gene) whose mutational site was poorly accessible, being part of a thermodynamically stable hairpin. In combination with use of auxiliary oligonucleotides (which restored the availability of each pre-defined hybridisation site), the assay demonstrated the ability to fully discriminate single-base mutations with detection limits in the high picomolar range (total analysis time: 60 min). Our specific probe design, hybridisation and signal transduction paths make the analytical process remarkably simple, relatively low cost and, thus, well suited for low throughput analysis of clinically relevant samples.
本文描述了一种基于使用分裂杂交探针的高选择性单核苷酸多态性（SNP）分型方法，并通过对实际患者样本中单个碱基突变的电化学分析来论证这一概念。要求两个探针彼此相邻杂交，以实现等位基因特异性寡核苷酸（ASO）的稳定化（通过碱基堆积）和结合，这为该检测赋予了高度严格的选择性标准。文中提供了调整此类堆积/ASO探针配对特性并在环境条件下实现完全错配区分（无需严格控制温度）的简单规则。所有基因分型实验均在室温下进行，使用一次性探针修饰金电极的平面表面作为基因传感平台。即使在大量单碱基取代序列存在的情况下仍能检测到纳摩尔量的合成靶标，有力地证明了分裂探针检测的特异性。为证明这种基因分型方法的普遍有效性，还进一步展示了该分析途径在临床靶标（从人TP53基因扩增而来）上的应用，这些靶标的突变位点难以接近，是热力学稳定发夹结构的一部分。结合使用辅助寡核苷酸（恢复每个预定义杂交位点的可用性），该检测方法展示了完全区分单碱基突变的能力，检测限在高皮摩尔范围内（总分析时间：60分钟）。我们独特的探针设计、杂交和信号转导路径使分析过程非常简单，成本相对较低，因此非常适合对临床相关样本进行低通量分析。
