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Multiplex PCR by multicolor fluorimetry and fluorescence melting curve analysis

Nature Medicine volume 7, pages 249–253 (2001)Cite this article

Sequence-specific fluorescent probe-based systems for the detection of nucleic acid sequences include those using linear hybridization probes1,2, TaqMan probes3, molecular beacons4,5,6 and peptide nucleic acid probes7. The linear hybridization probe format involves a fluorescence resonance energy transfer interaction8,9 between two fluorescently labeled oligonucleotide probes that hybridize adjacent to one another such that one fluorophore functions as the donor, while the other, the acceptor, emits light when juxtaposed to the donor1. This system is advantageous in that one probe may distinguish between products based on their distinct melting temperatures (Tms), which reflect the differences in the thermodynamic stabilities of the perfectly complementary and mismatched probe–target duplexes. Unfortunately, the number of products identifiable using this format is limited to those whose Tms can be distinguished10.

Fluorescence melting temperatures

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Figure 1: Probe orientation, spectral overlap and color compensation for multicolor Nras mutation detection.
Figure 2: Detection of homozygous point mutations in the Nras gene using a probe complementary to the wild-type Nras sequence.
Figure 3: Fluorescence melting peak analysis of homozygous codon 13 mutants.
Figure 4: Fluorescence melting peak analysis showing heterozygous Nras mutations.

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Acknowledgements

This work was supported by the ARUP Institute for Clinical and Experimental Pathology and by a Leukemia Research Foundation Grant to KSJE-J and National Institutes of Health Grants (CA83984) to KSJE-J and (GM58983) to CTW.

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Authors and Affiliations

  1. Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, USA

    Kojo S.J. Elenitoba-Johnson & Carl T. Wittwer

  2. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA

    Kojo S.J. Elenitoba-Johnson, Sandra D. Bohling & Carl T. Wittwer

  3. Lifespan Medical Center, and Brown University School of Medicine, Providence, Rhode Island, USA

    Thomas C. King

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Correspondence to Kojo S.J. Elenitoba-Johnson.

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Elenitoba-Johnson, K., Bohling, S., Wittwer, C. et al. Multiplex PCR by multicolor fluorimetry and fluorescence melting curve analysis. Nat Med 7, 249–253 (2001). https://doi.org/10.1038/84708

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