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Molecular Diagnostics and Genetics |
1 Department of Pathology, University of Utah Medical Center, 5B418, 50 N. Medical Dr., Salt Lake City, UT 84132.
aAuthor for correspondence. Fax 801-581-4517; e-mail carl.wittwer{at}path.utah.edu.
Background: Screening for heterozygous sequence changes in PCR products, also known as "mutation scanning", is an important tool for genetic research and clinical applications. Conventional methods require a separation step.
Methods: We evaluated the sensitivity and specificity of homogeneous scanning, using a saturating DNA dye and high-resolution melting. Heterozygous single-nucleotide polymorphism (SNP) detection was studied in three different sequence backgrounds of 40%, 50%, and 60% GC content. PCR products of 50–1000 bp were generated in the presence of LCGreenTM I. After fluorescence normalization and temperature overlay, melting curve shape was used to judge the presence or absence of heterozygotes among 1632 cases.
Results: For PCR products of 300 bp or less, all 280 heterozygous and 296 wild-type cases were correctly called without error. In 672 cases between 400 and 1000 bp with the mutation centered, the sensitivity and specificity were 96.1% and 99.4%, respectively. When the sequence background and product size with the greatest error rate were used, the sensitivity of off-center SNPs (384 cases) was 95.6% with a specificity of 99.4%. Most false negatives occurred with SNPs that were compared with an A or T wild type sequence.
Conclusions: High-resolution melting analysis with the dye LCGreen I identifies heterozygous single-base changes in PCR products with a sensitivity and specificity comparable or superior to nonhomogeneous techniques. The error rate of scanning depends on the PCR product size and the type of base change, but not on the position of the SNP. The technique requires only PCR reagents, the dye LCGreen I, and 1–2 min of closed-tube, post-PCR analysis.
The following articles in journals at HighWire Press have cited this article:
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  M.-P. Audrezet, A. Dabricot, C. Le Marechal, and C. Ferec Validation of High-Resolution DNA Melting Analysis for Mutation Scanning of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene J. Mol. Diagn., September 1, 2008; 10(5): 424 - 434. [Abstract] [Full Text] [PDF]
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 K. De Leeneer, I. Coene, B. Poppe, A. De Paepe, and K. Claes Rapid and Sensitive Detection of BRCA1/2 Mutations in a Diagnostic Setting: Comparison of Two High-Resolution Melting Platforms Clin. Chem., June 1, 2008; 54(6): 982 - 989. [Abstract] [Full Text] [PDF]
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 J.-H. Lin, C.-P. Tseng, Y.-J. Chen, C.-Y. Lin, S.-S. Chang, H.-S. Wu, and J.-C. Cheng Rapid Differentiation of Influenza A Virus Subtypes and Genetic Screening for Virus Variants by High-Resolution Melting Analysis J. Clin. Microbiol., March 1, 2008; 46(3): 1090 - 1097. [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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J. Montgomery, C. T. Wittwer, J. O. Kent, and L. Zhou Scanning the Cystic Fibrosis Transmembrane Conductance Regulator Gene Using High-Resolution DNA Melting Analysis Clin. Chem., November 1, 2007; 53(11): 1891 - 1898. [Abstract] [Full Text] [PDF]
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 A.-L. Chateigner-Boutin and I. Small A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons Nucleic Acids Res., September 27, 2007; 35(17): e114 - e114. [Abstract] [Full Text] [PDF]
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 N. Jeffery, R. B. Gasser, P. A. Steer, and A. H. Noormohammadi Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region Microbiology, August 1, 2007; 153(8): 2679 - 2688. [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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M. T. Seipp, J. D. Durtschi, M. A. Liew, J. Williams, K. Damjanovich, G. Pont-Kingdon, E. Lyon, K. V. Voelkerding, and C. T. Wittwer Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting J. Mol. Diagn., July 1, 2007; 9(3): 284 - 289. [Abstract] [Full Text] [PDF]
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J. G. Vandersteen, P. Bayrak-Toydemir, R. A. Palais, and C. T. Wittwer Identifying Common Genetic Variants by High-Resolution Melting Clin. Chem., July 1, 2007; 53(7): 1191 - 1198. [Abstract] [Full Text] [PDF]
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D. Fortini, A. Ciammaruconi, R. De Santis, A. Fasanella, A. Battisti, R. D'Amelio, F. Lista, A. Cassone, and A. Carattoli Optimization of High-Resolution Melting Analysis for Low-Cost and Rapid Screening of Allelic Variants of Bacillus anthracis by Multiple-Locus Variable-Number Tandem Repeat Analysis Clin. Chem., July 1, 2007; 53(7): 1377 - 1380. [Abstract] [Full Text] [PDF]
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R. L. Margraf, R. Mao, W. E. Highsmith, L. M. Holtegaard, and C. T. Wittwer RET Proto-Oncogene Genotyping Using Unlabeled Probes, the Masking Technique, and Amplicon High-Resolution Melting Analysis J. Mol. Diagn., April 1, 2007; 9(2): 184 - 196. [Abstract] [Full Text] [PDF]
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 J. B. Fiche, A. Buhot, R. Calemczuk, and T. Livache Temperature Effects on DNA Chip Experiments from Surface Plasmon Resonance Imaging: Isotherms and Melting Curves Biophys. J., February 1, 2007; 92(3): 935 - 946. [Abstract] [Full Text] [PDF]
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M. G. Herrmann, J. D. Durtschi, L. K. Bromley, C. T. Wittwer, and K. V. Voelkerding Instrument Comparison for Heterozygote Scanning of Single and Double Heterozygotes: A Correction and Extension of Herrmann et al., Clin Chem 2006;52:494-503 Clin. Chem., January 1, 2007; 53(1): 150 - 152. [Full Text] [PDF]
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 D. L. Faustman, S. D. Tran, S. Kodama, B. M. Lodde, I. Szalayova, S. Key, Z. E. Toth, and E. Mezey Comment on Papers by Chong et al., Nishio et al., and Suri et al. on Diabetes Reversal in NOD Mice Science, November 24, 2006; 314(5803): 1243a - 1243a. [Abstract] [Full Text] [PDF]
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J. Nishio, J. L. Gaglia, S. E. Turvey, C. Campbell, C. Benoist, and D. Mathis Response to Comment on Nishio et al. on Diabetes Reversal in NOD Mice Science, November 24, 2006; 314(5803): 1243c - 1243c. [Abstract] [Full Text] [PDF]
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U. Reischl Melting of the Ribosomal RNA Gene Reveals Bacterial Species Identity: A Step toward a New Rapid Test in Clinical Microbiology. Clin. Chem., November 1, 2006; 52(11): 1985 - 1987. [Full Text] [PDF]
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J.-C. Cheng, C.-L. Huang, C.-C. Lin, C.-C. Chen, Y.-C. Chang, S.-S. Chang, and C.-P. Tseng Rapid Detection and Identification of Clinically Important Bacteria by High-Resolution Melting Analysis after Broad-Range Ribosomal RNA Real-Time PCR Clin. Chem., November 1, 2006; 52(11): 1997 - 2004. [Abstract] [Full Text] [PDF]
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 C. G. Elliott, E. W. Glissmeyer, G. T. Havlena, J. Carlquist, J. T. McKinney, S. Rich, M. D. McGoon, M. B. Scholand, M. Kim, R. L. Jensen, et al. Relationship of BMPR2 Mutations to Vasoreactivity in Pulmonary Arterial Hypertension Circulation, May 30, 2006; 113(21): 2509 - 2515. [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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R. L. Margraf, R. Mao, W. E. Highsmith, L. M. Holtegaard, and C. T. Wittwer Mutation Scanning of the RET Protooncogene Using High-Resolution Melting Analysis Clin. Chem., January 1, 2006; 52(1): 138 - 141. [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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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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M. J. Smith, G. Pante-de-Sousa, K. K. Alharbi, X.-h. Chen, I. N.M. Day, and K. R. Fox Combination of His-Tagged T4 Endonuclease VII with Microplate Array Diagonal Gel Electrophoresis for High-Throughput Mutation Scanning Clin. Chem., June 1, 2005; 51(6): 1043 - 1046. [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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