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Department of Clinical Chemistry, Georg-August-University, Robert-Koch-Strasse 40, 37075 Goettingen, Germany.
a Author for correspondence. Fax 49-551-39-8551; e-mail nahsen{at}gwdg.de
Background: PCR-based mutation detection is prone to methodological errors, e.g., in restriction length fragment polymorphism (RFLP) and allele-specific amplification (ASA), false PCR results may occur because of technical faults or atypical new mutations.
Methods: We investigated the ability of a genotyping assay based on hybridization of labeled oligonucleotides to detect and discriminate known and as yet unknown mutations in the factor V and apolipoprotein B-100 genes. Expected melting points were calculated using a nearest-neighbor model for nucleic acid duplex stability and compared with experimental findings derived from LightCycler melting curves. A method for genotyping the apolipoprotein B-100 G10699A and C10698T mutations is presented.
Results: All mismatches tested for in the probed sequence could be detected with a single probe. The measured melting points were in good agreement with their values predicted using the nearest-neighbor model (r = 0.96; y = 0.98x + 1.18; Sy|x = 0.96; n = 24).
Conclusions: This procedure not only allows the identification of the mutation of interest but also enables the discrimination from other potential mutations in the vicinity of the former. The nearest-neighbor model is valid for hybridization probe assays on the LightCycler and should be of general value in setting up such assays. We have shown for two clinically relevant genotyping examples that the LightCycler mutation detection system has superior discriminatory performance compared with conventional RFLP or ASA PCR-based methods for molecular diagnostic purposes. With this method, in every hybridization probe assay, all mutations under a properly designed probe should be detectable, but they will not necessarily be discriminated from each other in all cases.
The following articles in journals at HighWire Press have cited this article:
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  R. P. Agarwal, S. M. Peters, M. Shemirani, and N. von Ahsen Improved Real-Time Multiplex Polymerase Chain Reaction Detection of Methylenetetrahydrofolate Reductase (MTHFR) 677C>T and 1298A>C Polymorphisms Using Nearest Neighbor Model-Based Probe Design J. Mol. Diagn., July 1, 2007; 9(3): 345 - 350. [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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 Z.-J. Tan and S.-J. Chen Nucleic Acid Helix Stability: Effects of Salt Concentration, Cation Valence and Size, and Chain Length Biophys. J., February 15, 2006; 90(4): 1175 - 1190. [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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 G. Pont-Kingdon and E. Lyon Direct molecular haplotyping by melting curve analysis of hybridization probes: beta 2-adrenergic receptor haplotypes as an example Nucleic Acids Res., June 3, 2005; 33(10): e89 - e89. [Abstract] [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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C. M. Strom, D. D. Clark, F. M. Hantash, L. Rea, B. Anderson, D. Maul, D. Huang, D. Traul, C. Chen Tubman, R. Garcia, et al. Direct Visualization of Cystic Fibrosis Transmembrane Regulator Mutations in the Clinical Laboratory Setting Clin. Chem., May 1, 2004; 50(5): 836 - 845. [Abstract] [Full Text] [PDF]
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E. Dempsey, D. E. Barton, and F. Ryan Detection of Five Common CFTR Mutations by Rapid-Cycle Real-Time Amplification Refractory Mutation System PCR Clin. Chem., April 1, 2004; 50(4): 773 - 775. [Full Text] [PDF]
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N. von Ahsen Labeled Primers for Mutation Scanning: Making Diagnostic Use of the Nucleobase Quenching Effect Clin. Chem., March 1, 2003; 49(3): 355 - 356. [Full Text] [PDF]
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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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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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 M. Davis, R. Brown, A. Dickson, H. Horton, D. James, N. Laing, R. Marston, M. Norgate, D. Perlman, N. Pollock, et al. Malignant hyperthermia associated with exercise-induced rhabdomyolysis or congenital abnormalities and a novel RYR1 mutation in New Zealand and Australian pedigrees Br. J. Anaesth., April 1, 2002; 88(4): 508 - 515. [Abstract] [Full Text] [PDF]
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N. von Ahsen, C. T. Wittwer, and E. Schutz Oligonucleotide Melting Temperatures under PCR Conditions: Nearest-Neighbor Corrections for Mg2+, Deoxynucleotide Triphosphate, and Dimethyl Sulfoxide Concentrations with Comparison to Alternative Empirical Formulas Clin. Chem., November 1, 2001; 47(11): 1956 - 1961. [Abstract] [Full Text] [PDF]
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M. Orth, S. Westphal, J. Dierkes, C. Luley, and K. Schlatterer Rapid Factor XII (46C{->}T) Genotyping by Fluorescence Resonance Energy Transfer in Patients with Coronary Artery Disease or Thrombophilia Clin. Chem., June 1, 2001; 47(6): 1117 - 1119. [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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N. von Ahsen, M. Oellerich, and E. Schutz DNA Base Bulge vs Unmatched End Formation in Probe-based Diagnostic Insertion/Deletion Genotyping: Genotyping the UGT1A1 (TA)n Polymorphism by Real-Time Fluorescence PCR Clin. Chem., December 1, 2000; 46(12): 1939 - 1945. [Abstract] [Full Text] [PDF]
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M. Nauck, U. Stein, S. von Karger, W. Marz, and H. Wieland Rapid Detection of the C3435T Polymorphism of Multidrug Resistance Gene 1 Using Fluorogenic Hybridization Probes Clin. Chem., December 1, 2000; 46(12): 1995 - 1997. [Full Text] [PDF]
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E. Schutz, N. von Ahsen, and M. Oellerich Genotyping of Eight Thiopurine Methyltransferase Mutations: Three-Color Multiplexing, ""Two-Color/Shared"" Anchor, and Fluorescence-quenching Hybridization Probe Assays Based on Thermodynamic Nearest-Neighbor Probe Design Clin. Chem., November 1, 2000; 46(11): 1728 - 1737. [Abstract] [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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