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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 20–45 min and requires no sample manipulation after the fluorescence thermal cycler is loaded.
The following articles in journals at HighWire Press have cited this article:
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  C. N. Gundry, S. F. Dobrowolski, Y. R. Martin, T. C. Robbins, L. M. Nay, N. Boyd, T. Coyne, M. D. Wall, C. T. Wittwer, and D. H.-F. Teng Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons Nucleic Acids Res., June 1, 2008; 36(10): 3401 - 3408. [Abstract] [Full Text] [PDF]
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 M. T. Seipp, D. Pattison, J. D. Durtschi, M. Jama, K. V. Voelkerding, and C. T. Wittwer Quadruplex Genotyping of F5, F2, and MTHFR Variants in a Single Closed Tube by High-Resolution Amplicon Melting Clin. Chem., January 1, 2008; 54(1): 108 - 115. [Abstract] [Full Text] [PDF]
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M. G. Herrmann, J. D. Durtschi, C. T. Wittwer, and K. V. Voelkerding Expanded Instrument Comparison of Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping Clin. Chem., August 1, 2007; 53(8): 1544 - 1548. [Abstract] [Full Text] [PDF]
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E. A. Lukhtanov, S. G. Lokhov, V. V. Gorn, M. A. Podyminogin, and W. Mahoney Novel DNA probes with low background and high hybridization-triggered fluorescence Nucleic Acids Res., March 12, 2007; 35(5): e30 - e30. [Abstract] [Full Text] [PDF]
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 L. Stromqvist Meuzelaar, K. Hopkins, E. Liebana, and A. J. Brookes DNA Diagnostics by Surface-Bound Melt-Curve Reactions J. Mol. Diagn., February 1, 2007; 9(1): 30 - 41. [Abstract] [Full Text] [PDF]
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E. Schutz, M. Scharfenstein, and B. Brenig Genotyping of Ovine Prion Protein Gene (PRNP) Variants by PCR with Melting Curve Analysis, Clin. Chem., July 1, 2006; 52(7): 1426 - 1429. [Abstract] [Full Text] [PDF]
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M. G. Herrmann, J. D. Durtschi, L. K. Bromley, C. T. Wittwer, and K. V. Voelkerding Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping: Cross-Platform Comparison of Instruments and Dyes Clin. Chem., March 1, 2006; 52(3): 494 - 503. [Abstract] [Full Text] [PDF]
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N. von Ahsen Two for Typing: Homogeneous Combined Single-Nucleotide Polymorphism Scanning and Genotyping Clin. Chem., October 1, 2005; 51(10): 1761 - 1762. [Full Text] [PDF]
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L. Zhou, L. Wang, R. Palais, R. Pryor, and C. T. Wittwer High-Resolution DNA Melting Analysis for Simultaneous Mutation Scanning and Genotyping in Solution Clin. Chem., October 1, 2005; 51(10): 1770 - 1777. [Abstract] [Full Text] [PDF]
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M. S. Mahadevan and P. V. Benson Factor V Null Mutation Affecting the Roche LightCycler Factor V Leiden Assay Clin. Chem., August 1, 2005; 51(8): 1533 - 1535. [Full Text] [PDF]
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F. Frances, D. Corella, J. V. Sorli, M. Guillen, J. I. Gonzalez, and O. Portoles Validating a Rapid Method for Detecting Common Polymorphisms in the APOA5 Gene by Melting Curve Analysis Using LightTyper Clin. Chem., July 1, 2005; 51(7): 1279 - 1282. [Full Text] [PDF]
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A. Verri, F. Focher, G. Tettamanti, and V. Grazioli Two-Step Genetic Screening of Thrombophilia by Pyrosequencing Clin. Chem., July 1, 2005; 51(7): 1282 - 1284. [Full Text] [PDF]
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R. Graham, M. Liew, C. Meadows, E. Lyon, and C. T. Wittwer Distinguishing Different DNA Heterozygotes by High-Resolution Melting Clin. Chem., July 1, 2005; 51(7): 1295 - 1298. [Full Text] [PDF]
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 K. E. Pierce, J. A. Sanchez, J. E. Rice, and L. J. Wangh Linear-After-The-Exponential (LATE)-PCR: Primer design criteria for high yields of specific single-stranded DNA and improved real-time detection PNAS, June 14, 2005; 102(24): 8609 - 8614. [Abstract] [Full Text] [PDF]
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T. R. Gingeras, R. Higuchi, L. J. Kricka, Y.M. D. Lo, and C. T. Wittwer Fifty Years of Molecular (DNA/RNA) Diagnostics Clin. Chem., March 1, 2005; 51(3): 661 - 671. [Full Text] [PDF]
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M. Berger, S. Zschemisch, B. Hocher, D. Monks, T. Slowinski, H. Halle, C. Wanner, U. Walter, and T. H. Lindner Alternative Approach for Rapid and Reliable Single-Nucleotide Polymorphism Typing with Double Restriction Mutagenesis Primer PCR Clin. Chem., December 1, 2004; 50(12): 2376 - 2378. [Full Text] [PDF]
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R. Rej Clinical Chemistry through Clinical Chemistry: A Journal Timeline Clin. Chem., December 1, 2004; 50(12): 2415 - 2458. [Abstract] [Full Text] [PDF]
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L. A. Ugozzoli Multiplex Assays with Fluorescent Microbead Readout: A Powerful Tool for Mutation Detection Clin. Chem., November 1, 2004; 50(11): 1963 - 1965. [Full Text] [PDF]
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G. H. Reed and C. T. Wittwer Sensitivity and Specificity of Single-Nucleotide Polymorphism Scanning by High-Resolution Melting Analysis Clin. Chem., October 1, 2004; 50(10): 1748 - 1754. [Abstract] [Full Text] [PDF]
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H. Gorgens, G. Fitze, D. Roesner, and H. K. Schackert One-Step Analysis of Ten Functional Haplotype Combinations of the Basic RET Promoter with a LightCycler Assay Clin. Chem., September 1, 2004; 50(9): 1693 - 1695. [Full Text] [PDF]
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L. Zhou, A. N. Myers, J. G. Vandersteen, L. Wang, and C. T. Wittwer Closed-Tube Genotyping with Unlabeled Oligonucleotide Probes and a Saturating DNA Dye Clin. Chem., August 1, 2004; 50(8): 1328 - 1335. [Abstract] [Full Text] [PDF]
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M. Liew, R. Pryor, R. Palais, C. Meadows, M. Erali, E. Lyon, and C. Wittwer Genotyping of Single-Nucleotide Polymorphisms by High-Resolution Melting of Small Amplicons Clin. Chem., July 1, 2004; 50(7): 1156 - 1164. [Abstract] [Full Text] [PDF]
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R. Luderer, A. Verheul, and W. Kortlandt Rapid Detection of the Factor V Leiden Mutation by Real-Time PCR with TaqMan Minor Groove Binder Probes Clin. Chem., April 1, 2004; 50(4): 787 - 788. [Full Text] [PDF]
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M. Panning, M. Asper, S. Kramme, H. Schmitz, and C. Drosten Rapid Detection and Differentiation of Human Pathogenic Orthopox Viruses by a Fluorescence Resonance Energy Transfer Real-Time PCR Assay Clin. Chem., April 1, 2004; 50(4): 702 - 708. [Abstract] [Full Text] [PDF]
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 R. Selvarangan, U. Bui, A. P. Limaye, and B. T. Cookson Rapid Identification of Commonly Encountered Candida Species Directly from Blood Culture Bottles J. Clin. Microbiol., December 1, 2003; 41(12): 5660 - 5664. [Abstract] [Full Text] [PDF]
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A. Millson, A. Suli, L. Hartung, S. Kunitake, A. Bennett, M. C. L. Nordberg, W. Hanna, C. T. Wittwer, A. Seth, and E. Lyon Comparison of Two Quantitative Polymerase Chain Reaction Methods for Detecting HER2/neu Amplification J. Mol. Diagn., August 1, 2003; 5(3): 184 - 190. [Abstract] [Full Text] [PDF]
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H. Gorgens, P. Schwarz, J. Schulze, and H. K. Schackert LightCycler Assay in the Analysis of Haplotypes of the Type 2 Diabetes Susceptibility Gene CAPN10 Clin. Chem., August 1, 2003; 49(8): 1405 - 1408. [Full Text] [PDF]
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 C. P. Vaughn and K. S.J. Elenitoba-Johnson Hybridization-Induced Dequenching of Fluorescein-Labeled Oligonucleotides: A Novel Strategy for PCR Detection and Genotyping Am. J. Pathol., July 1, 2003; 163(1): 29 - 35. [Abstract] [Full Text] [PDF]
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C. T. Wittwer, G. H. Reed, C. N. Gundry, J. G. Vandersteen, and R. J. Pryor High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen Clin. Chem., June 1, 2003; 49(6): 853 - 860. [Abstract] [Full Text] [PDF]
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 M. Jobs, W. M. Howell, L. Stromqvist, T. Mayr, and A. J. Brookes DASH-2: Flexible, Low-Cost, and High-Throughput SNP Genotyping by Dynamic Allele-Specific Hybridization on Membrane Arrays Genome Res., May 1, 2003; 13(5): 916 - 924. [Abstract] [Full Text] [PDF]
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M. Erali, B. Schmidt, E. Lyon, and C. Wittwer Evaluation of Electronic Microarrays for Genotyping Factor V, Factor II, and MTHFR Clin. Chem., May 1, 2003; 49(5): 732 - 739. [Abstract] [Full Text] [PDF]
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A. Schulz, K. Mellenthin, G. Schonian, B. Fleischer, and C. Drosten Detection, Differentiation, and Quantitation of Pathogenic Leishmania Organisms by a Fluorescence Resonance Energy Transfer-Based Real-Time PCR Assay J. Clin. Microbiol., April 1, 2003; 41(4): 1529 - 1535. [Abstract] [Full Text] [PDF]
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S. F. Dobrowolski, R. A. Banas, J. G. Suzow, M. Berkley, and E. W. Naylor Analysis of Common Mutations in the Galactose-1-Phosphate Uridyl Transferase Gene: New Assays to Increase the Sensitivity and Specificity of Newborn Screening for Galactosemia J. Mol. Diagn., February 1, 2003; 5(1): 42 - 47. [Abstract] [Full Text] [PDF]
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 F. Rodriguez, R. Jardi, X. Costa, M. Cotrina, R. Galimany, R. Vidal, and M. Miravitlles Rapid Screening for {alpha}1-Antitrypsin Deficiency in Patients with Chronic Obstructive Pulmonary Disease Using Dried Blood Specimens Am. J. Respir. Crit. Care Med., September 15, 2002; 166(6): 814 - 817. [Abstract] [Full Text]
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S. M. Williams, C. A. Meadows, and E. Lyon Automated DNA Extraction for Real-Time PCR Clin. Chem., September 1, 2002; 48(9): 1629 - 1630. [Full Text] [PDF]
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P. S. Bernard and C. T. Wittwer Real-Time PCR Technology for Cancer Diagnostics Clin. Chem., August 1, 2002; 48(8): 1178 - 1185. [Abstract] [Full Text] [PDF]
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H. Millward, W. Samowitz, C. T. Wittwer, and P. S. Bernard Homogeneous Amplification and Mutation Scanning of the p53 Gene Using Fluorescent Melting Curves Clin. Chem., August 1, 2002; 48(8): 1321 - 1328. [Abstract] [Full Text] [PDF]
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I. M. Mackay, K. E. Arden, and A. Nitsche Real-time PCR in virology Nucleic Acids Res., March 15, 2002; 30(6): 1292 - 1305. [Abstract] [Full Text] [PDF]
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S. Burggraf, S. Kosel, S. Lohmann, R. Beck, and B. Olgemoller Unexplained DNA Melting Behavior in a Genotyping Assay Clin. Chem., January 1, 2002; 48(1): 199 - 201. [Full Text] [PDF]
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F. R. Stamey, M. M. Patel, B. P. Holloway, and P. E. Pellett Quantitative, Fluorogenic Probe PCR Assay for Detection of Human Herpesvirus 8 DNA in Clinical Specimens J. Clin. Microbiol., October 1, 2001; 39(10): 3537 - 3540. [Abstract] [Full Text] [PDF]
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A. Vega, C. Ruiz-Ponte, A. Carracedo, and F. Barros Rapid Genotyping of the M129V Polymorphism of Prion Protein Using Real-Time Fluorescent PCR Clin. Chem., October 1, 2001; 47(10): 1874 - 1875. [Full Text] [PDF]
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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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E. Lyon, A. Millson, M. C. Lowery, R. Woods, and C. T. Wittwer Quantification of HER2/neu Gene Amplification by Competitive PCR Using Fluorescent Melting Curve Analysis Clin. Chem., May 1, 2001; 47(5): 844 - 851. [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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L. M. Real, A. J. Gayoso, M. Olivera, A. Caruz, A. Ruiz, and F. Gayoso Detection of Nucleotide c985 A{->}G Mutation of Medium-Chain Acyl-CoA Dehydrogenase Gene by Real-Time PCR Clin. Chem., May 1, 2001; 47(5): 958 - 959. [Full Text] [PDF]
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A. A. Reyes, L. A. Ugozzoli, J. D. Lowery, J. W. Breneman III, C. S. Hixson, R. D. Press, and R. B. Wallace Linked Linear Amplification: A New Method for the Amplification of DNA Clin. Chem., January 1, 2001; 47(1): 31 - 40. [Abstract] [Full Text] [PDF]
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F. A.J.T.M. van den Bergh, A. M. van Oeveren-Dybicz, and M. A.M. Bon Rapid Single-Tube Genotyping of the Factor V Leiden and Prothrombin Mutations by Real-Time PCR Using Dual-Color Detection Clin. Chem., August 1, 2000; 46(8): 1191 - 1195. [Full Text] [PDF]
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M. A.M. Bon, A. van Oeveren-Dybicz, and F. A.J.T.M van den Bergh Genotyping of HLA-B27 by Real-Time PCR without Hybridization Probes Clin. Chem., July 1, 2000; 46(7): 1000 - 1002. [Full Text] [PDF]
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J. Loeffler, L. Hagmeyer, H. Hebart, N. Henke, U. Schumacher, and H. Einsele Rapid Detection of Point Mutations by Fluorescence Resonance Energy Transfer and Probe Melting Curves in Candida Species Clin. Chem., May 1, 2000; 46(5): 631 - 635. [Abstract] [Full Text] [PDF]
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M. Nauck, M. M. Hoffmann, H. Wieland, and W. Marz Evaluation of the Apo E Genotyping Kit on the LightCycler, Clin. Chem., May 1, 2000; 46(5): 722 - 724. [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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N. von Ahsen, M. Oellerich, and E. Schutz Use of Two Reporter Dyes without Interference in a Single-Tube Rapid-Cycle PCR: {alpha}1-Antitrypsin Genotyping by Multiplex Real-Time Fluorescence PCR with the LightCycler Clin. Chem., February 1, 2000; 46(2): 156 - 161. [Abstract] [Full Text] [PDF]
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T. Aoshima, Y. Sekido, T. Miyazaki, M. Kajita, S. Mimura, K. Watanabe, K. Shimokata, and T. Niwa Rapid Detection of Deletion Mutations in Inherited Metabolic Diseases by Melting Curve Analysis with LightCycler Clin. Chem., January 1, 2000; 46(1): 119 - 122. [Full Text] [PDF]
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N. von Ahsen, M. Oellerich, V. W. Armstrong, and E. Schutz Application of a Thermodynamic Nearest-Neighbor Model to Estimate Nucleic Acid Stability and Optimize Probe Design: Prediction of Melting Points of Multiple Mutations of Apolipoprotein B-3500 and Factor V with a Hybridization Probe Genotyping Assay on the LightCycler Clin. Chem., December 1, 1999; 45(12): 2094 - 2101. [Abstract] [Full Text] [PDF]
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C. Aslanidis, M. Nauck, and G. Schmitz High-Speed Detection of the Two Common {alpha}1-Antitrypsin Deficiency Alleles Pi*Z and Pi*S by Real-Time Fluorescence PCR and Melting Curves Clin. Chem., October 1, 1999; 45(10): 1872 - 1875. [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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