Wilson P K, Jiang T, Minunni M E, Turner A P F, Mascini M
Cranfield University, UK.
Biosens Bioelectron. 2005 May 15;20(11):2310-3. doi: 10.1016/j.bios.2004.11.020.
The TP53 gene has been the subject of intense research since the realisation that inactivation of this gene is common to most cancer types. Numerous publications have linked TP53 mutations in general or at specific locations to patient prognosis and therapy response. The findings of many studies using general approaches such as immunohistochemistry or sequencing are contradictory. However, the detection of specific mutations, especially those occurring in the structurally important L2 and L3 zinc binding domains, which are the most common sites of TP53 mutations, have been linked to patient prognosis and more strongly to radiotherapy and chemotherapy resistance in several major cancers. In this study, the TI-SPR-1 surface plasmon resonance system and Texas Instruments Spreeta chips were used to develop a DNA biosensor based on thiolated probes complementary to these domains. The sensors were able to detect these mutations in both oligonucleotides and PCR products with normal and mutant TP53 DNA, but the difference in hybridisation signal was small. Preliminary experiments to enhance the signal using Escherichia coli mismatch repair proteins, MutS and single strand binding protein were carried out. It was found that MutS was unable to bind to mismatch oligonucleotides, but single strand binding protein was able to bind to single stranded probes, which had not hybridised to the target, resulting in a three-fold increase in the sensitivity of the biosensor. While further work needs to be carried out to optimise the system, these preliminary experiments indicate that the TI-SPR-1 can be used for the detection of clinically relevant mutations in the TP53 gene and that the sensitivity can be increased significantly using single strand binding protein. This system has a number of advantages over current mutation detection technologies, including lower cost, ease of sensor preparation and measurement procedures, technical simplicity and increased speed due to the lack of need for gel electrophoresis.
自从认识到该基因失活在大多数癌症类型中都很常见以来，TP53基因一直是深入研究的对象。众多出版物将一般或特定位置的TP53突变与患者预后及治疗反应联系起来。许多使用免疫组织化学或测序等一般方法的研究结果相互矛盾。然而，特定突变的检测，尤其是那些发生在结构重要的L2和L3锌结合结构域中的突变，这些结构域是TP53突变最常见的位点，已在几种主要癌症中与患者预后相关联，并且与放疗和化疗耐药性的关联更为紧密。在本研究中，使用TI-SPR-1表面等离子体共振系统和德州仪器Spreeta芯片开发了一种基于与这些结构域互补的硫醇化探针的DNA生物传感器。这些传感器能够在含有正常和突变TP53 DNA的寡核苷酸和PCR产物中检测到这些突变，但杂交信号的差异很小。进行了使用大肠杆菌错配修复蛋白MutS和单链结合蛋白增强信号的初步实验。发现MutS无法与错配寡核苷酸结合，但单链结合蛋白能够与未与靶标杂交的单链探针结合，从而使生物传感器的灵敏度提高了三倍。虽然需要进一步开展工作来优化该系统，但这些初步实验表明，TI-SPR-1可用于检测TP53基因中的临床相关突变，并且使用单链结合蛋白可显著提高灵敏度。该系统相对于当前的突变检测技术具有许多优势，包括成本更低、传感器制备和测量程序简便、技术简单以及由于无需凝胶电泳而提高了速度。
