Abstract
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.
Highlights
High throughput sequencing approaches have facilitated genome-wide discovery of mutations characteristic of various disease statuses [1]
The results showed that, as expected, fluorescence intensity of the hybrids decreased as temperature increased in a target-dependant manner (Figure 1A, middle panel), yielding different Tm value for each target derived from the melting peak (Figure 1A, bottom panel and Table S1). These results demonstrated the feasibility of TaqMan probe for fluorescence melting curve analysis (FMCA)
DNA sequencing result revealed a triple mutation (530 CTG.ATG/531 TCG.TTC), a variation not found previously among the local patients. These results demonstrate that TaqMan probe-based FMCA with color multiplexing can be directly used to scan the existence of unknown mutations within the amplicon
Summary
High throughput sequencing approaches have facilitated genome-wide discovery of mutations characteristic of various disease statuses [1]. Since each mutation needs a specific probe with a unique color, the number of distinguishable fluorophores and fluorescence detection channels in a fluorometric thermocycler becomes the bottleneck for multiplex detection. These limitations can be addressed by a post-PCR, probe-based fluorescence melting curve analysis (FMCA) procedure that allows detection of multiple mutations by a single probe based on melting temperature (Tm) shifts [6]. FMCA has become a versatile tool for mutation detection [10,11]
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