title: Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes.
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.
authors.affiliation: 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.
authors.forename: Qiuying
authors.initials: Q
authors.lastname: Huang
authors.forename: Zanzan
authors.initials: Z
authors.lastname: Liu
authors.forename: Yiqun
authors.initials: Y
authors.lastname: Liao
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Chen
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Qingge
authors.initials: Q
authors.lastname: Li
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003116:Color; D004252:DNA Mutational Analysis / Q000379:methods*; D012321:DNA-Directed RNA Polymerases; D005838:Genotype; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D019914:Taq Polymerase / Q000378:metabolism; D044366:Transition Temperature
chemical_list: D001426:Bacterial Proteins; D015345:Oligonucleotide Probes; C505805:rpoB protein, Mycobacterium tuberculosis; D019914:Taq Polymerase; D012321:DNA-Directed RNA Polymerases