Isacsson J, Cao H, Ohlsson L, Nordgren S, Svanvik N, Westman G, Kubista M, Sjöback R, Sehlstedt U
Department of Molecular Biotechnology, Chalmers University of Technology, S-405 30 Göteborg, Sweden.
Mol Cell Probes. 2000 Oct;14(5):321-8. doi: 10.1006/mcpr.2000.0321.
Newly developed light-up probes offer an attractive tool for PCR product detection. The light-up probe, which consists of a thiazole orange derivative linked to a peptide nucleic acid oligomer, hybridizes specifically to complementary nucleic acids. Upon hybridization the thiazole orange moiety interacts with the nucleic acid bases and the probe becomes brightly fluorescent. This eliminates the need to separate bound from unbound probes and reduces the risk of cross contamination during sample handling. We demonstrate here the applicability of light-up probes in two different PCR assays, one directed towards the human beta-actin gene and the other towards the invA gene of Salmonella. The probes do not interfere with the PCR reaction and can either be included in the sample mixture or added after completed amplification. The specificity of the probe is found to be excellent: a single-base mismatch in the target sequence is sufficient to prevent probe binding as indicated by the lack of fluorescence increase. Furthermore, a clear correlation is found between the intensity of gel bands and the measured probe fluorescence in solution, which suggests that the amount of PCR products can be quantified using light-up probes.
新开发的发光探针为PCR产物检测提供了一种有吸引力的工具。这种发光探针由与肽核酸寡聚物相连的噻唑橙衍生物组成，能与互补核酸特异性杂交。杂交后，噻唑橙部分与核酸碱基相互作用，探针变得发出明亮荧光。这消除了分离结合探针与未结合探针的需要，并降低了样品处理过程中交叉污染的风险。我们在此展示了发光探针在两种不同PCR检测中的适用性，一种针对人类β-肌动蛋白基因，另一种针对沙门氏菌的invA基因。这些探针不干扰PCR反应，可以包含在样品混合物中或在扩增完成后添加。发现探针的特异性极佳：靶序列中的单个碱基错配足以阻止探针结合，如荧光没有增加所示。此外，在凝胶条带强度与溶液中测得的探针荧光之间发现了明显的相关性，这表明可以使用发光探针定量PCR产物的量。
