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Articles |
1
ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108.
2
Department of Pathology, University of Utah Medical
Center, 50 N. Medical Dr., Salt Lake City, UT 84132. Fax 801-581-4517;
e-mail Carl_Wittwer{at}hlthsci.med.utah.edu
a Author for correspondence.
A single-step method for factor V Leiden genotyping is presented that uses rapid-cycle PCR and simultaneous fluorescence analysis with resonance energy transfer probes. A fragment of the factor V gene containing the mutation is amplified asymmetrically through use of a primer labeled with Cy5TM in the presence of a 3'-fluorescein-labeled probe that covers the mutation site. When the fluorescein probe is annealed to the extension product of the Cy5-labeled primer, the fluorophores are brought into close enough contact for resonance energy transfer to occur. As the temperature increases, the probe melts from its target, decreasing the resonance energy transfer. When the probe is complementary to the product strand, it melts at 65 °C; if the single-base mutation is present, the probe melts at 57 °C. Concurrent amplification and analysis from genomic DNA takes 2045 min and requires no sample manipulation after the fluorescence thermal cycler is loaded.
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C. A. Foy and H. C. Parkes Emerging Homogeneous DNA-based Technologies in the Clinical Laboratory Clin. Chem., June 1, 2001; 47(6): 990 - 1000. [Abstract] [Full Text] [PDF] |
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D. Teupser, W. Rupprecht, P. Lohse, and J. Thiery Fluorescence-based Detection of the CETP TaqIB Polymorphism: False Positives with the TaqMan-based Exonuclease Assay Attributable to a Previously Unknown Gene Variant Clin. Chem., May 1, 2001; 47(5): 852 - 857. [Abstract] [Full Text] [PDF] |
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M. G. Herrmann, S. F. Dobrowolski, and C. T. Wittwer Rapid {beta}-Globin Genotyping by Multiplexing Probe Melting Temperature and Color Clin. Chem., March 1, 2000; 46(3): 425 - 428. [Full Text] [PDF] |
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P. S. Bernard and C. T. Wittwer Homogeneous Amplification and Variant Detection by Fluorescent Hybridization Probes Clin. Chem., February 1, 2000; 46(2): 147 - 148. [Full Text] [PDF] |
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K. Mangasser-Stephan, C. Tag, A. Reiser, and A. M. Gressner Rapid Genotyping of Hemochromatosis Gene Mutations on the LightCycler with Fluorescent Hybridization Probes Clin. Chem., October 1, 1999; 45(10): 1875 - 1878. [Full Text] [PDF] |
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C. Aslanidis and G. Schmitz High-Speed Apolipoprotein E Genotyping and Apolipoprotein B3500 Mutation Detection Using Real-Time Fluorescence PCR and Melting Curves Clin. Chem., July 1, 1999; 45(7): 1094 - 1097. [Full Text] [PDF] |
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N. von Ahsen, E. Schutz, V. W. Armstrong, and M. Oellerich Rapid Detection of Prothrombotic Mutations of Prothrombin (G20210A), Factor V (G1691A), and Methylenetetrahydrofolate Reductase (C677T) by Real-Time Fluorescence PCR with the LightCycler Clin. Chem., May 1, 1999; 45(5): 694 - 696. [Full Text] [PDF] |
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P. S. Bernard, R. S. Ajioka, J. P. Kushner, and C. T. Wittwer Homogeneous Multiplex Genotyping of Hemochromatosis Mutations with Fluorescent Hybridization Probes Am. J. Pathol., October 1, 1998; 153(4): 1055 - 1061. [Abstract] [Full Text] [PDF] |
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