Engineering Research Center of Molecular Diagnostics, Ministry of Education, Department of Biomedical Sciences, School of Life Sciences, and Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, Xiamen University, Xiamen, Fujian, China.
PLoS One. 2011 Apr 28;6(4):e19206. doi: 10.1371/journal.pone.0019206.
Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.
基于探针的荧光熔解曲线分析（FMCA）是一种强大的突变检测工具，基于探针-靶标杂交体热变性产生的熔解温度。然而，现有的探针化学性质限制了其在颜色多重性、探针设计和跨平台兼容性方面的发展。我们在此探索了两种双重标记的、自淬灭探针，即 TaqMan 和共享茎分子信标，以评估它们在 FMCA 中的应用能力。这两种探针都可以直接用于 FMCA，并在不对称 PCR 条件下与封闭管扩增杂交轻松集成。通过使用这些探针，FMCA 的灵活性得到了显著提高，在 FMCA 的三个代表性应用中得到了验证：突变扫描、突变鉴定和突变基因分型，所有这些应用都实现了颜色多重性的改善，探针设计更加简单，探针组合更加多样，并且都经过了大量真实临床样本的验证。这些基于探针的 FMCA 具有通用的跨平台兼容性，在五种不同的实时 PCR 仪器上进行的 4 色突变基因分型检测中得到了验证。双重标记的、自淬灭探针提供了前所未有的优势，包括增强的多重性、探针设计的灵活性提高以及跨平台兼容性的扩展，这将极大地改善各种应用中的突变检测中的 FMCA。
