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Received on March 16, 2004
Accepted on April 30, 2004
Molecular Diagnostics and Genetics |
1 Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT
* To whom correspondence should be addressed. E-mail: carl.wittwer{at}path.utah.edu.
Background: Homogeneous PCR methods for genotyping usually require fluorescently labeled oligonucleotide probes. Amplicon melting with the DNA dye LCGreenTM I was recently introduced as a closed-tube method of genotyping that does not require probes or real-time PCR. However, some single-nucleotide polymorphisms (SNPs) could not be completely genotyped without addition of a known genotype, and high-resolution melting techniques were necessary.
Methods: A 3'-blocked, unlabeled oligonucleotide probe and the saturating dye, LCGreen I, were added to standard PCR reagents before amplification. After PCR, the samples were melted at 0.1-0.3 °C/s in high-resolution (HR-1TM), high-throughput (LightTyperTM) and rapid-cycle, real-time (LightCycler®) instruments, and fluorescence melting curves were recorded.
Results: Derivative melting curves of the probe-target duplexes were characteristic of the genotype under the probe. With synthetic plasmid templates, all SNP base combinations could be genotyped. For human genomic DNA, the technique was demonstrated with mutations associated with cystic fibrosis, including SNPs (G542X, I506V, and F508C) and 3-bp deletions (F508del and I507del).
Conclusions: Genotyping of SNPs and small deletions by melting analysis of an unlabeled probe in the presence of LCGreen I is simple and rapid. Only three unlabeled oligonucleotides (two primers and one probe), a saturating DNA dye, PCR, and a melting instrument are required. The method is closed-tube, does not require fluorescently labeled probes or real-time PCR, and can be completed in <10 min on any instrument capable of monitoring melting curves by fluorescence.
The following articles in journals at HighWire Press have cited this article:
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  K. De Leeneer, I. Coene, B. Poppe, A. De Paepe, and K. Claes Genotyping of Frequent BRCA1/2 SNPs with Unlabeled Probes: A Supplement to HRMCA Mutation Scanning, Allowing the Strong Reduction of Sequencing Burden J. Mol. Diagn., September 1, 2009; 11(5): 415 - 419. [Abstract] [Full Text] [PDF]
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E. Lyon and C. T. Wittwer LightCycler Technology in Molecular Diagnostics J. Mol. Diagn., March 1, 2009; 11(2): 93 - 101. [Abstract] [Full Text] [PDF]
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 L. Zhou, R. J. Errigo, H. Lu, M. A. Poritz, M. T. Seipp, and C. T. Wittwer Snapback Primer Genotyping with Saturating DNA Dye and Melting Analysis Clin. Chem., October 1, 2008; 54(10): 1648 - 1656. [Abstract] [Full Text] [PDF]
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T. S. Laughlin, M. W. Becker, J. L. Liesveld, D. A. Mulford, C. N. Abboud, P. Brown, and P. G. Rothberg Rapid Method for Detection of Mutations in the Nucleophosmin Gene in Acute Myeloid Leukemia J. Mol. Diagn., July 1, 2008; 10(4): 338 - 345. [Abstract] [Full Text] [PDF]
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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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G. Pont-Kingdon, R. L. Margraf, K. Sumner, A. Millson, E. Lyon, and E. Schutz Design and Application of Noncontinuously Binding Probes Used for Haplotyping and Genotyping Clin. Chem., June 1, 2008; 54(6): 990 - 999. [Abstract] [Full Text] [PDF]
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R. L. Margraf, R. Mao, and C. T. Wittwer Rapid Diagnosis of MEN2B Using Unlabeled Probe Melting Analysis and the LightCycler 480 Instrument J. Mol. Diagn., March 1, 2008; 10(2): 123 - 128. [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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S. Dames, D. C. Pattison, L. K. Bromley, C. T. Wittwer, and K. V. Voelkerding Unlabeled Probes for the Detection and Typing of Herpes Simplex Virus Clin. Chem., October 1, 2007; 53(10): 1847 - 1854. [Abstract] [Full Text] [PDF]
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S. Dames, R. L. Margraf, D. C. Pattison, C. T. Wittwer, and K. V. Voelkerding Characterization of Aberrant Melting Peaks in Unlabeled Probe Assays J. Mol. Diagn., July 1, 2007; 9(3): 290 - 296. [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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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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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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M. Liew, L. Nelson, R. Margraf, S. Mitchell, M. Erali, R. Mao, E. Lyon, and C. Wittwer Genotyping of Human Platelet Antigens 1 to 6 and 15 by High-Resolution Amplicon Melting and Conventional Hybridization Probes J. Mol. Diagn., February 1, 2006; 8(1): 97 - 104. [Abstract] [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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E. Lyon Discovering Rare Variants by Use of Melting Temperature Shifts Seen in Melting Curve Analysis Clin. Chem., August 1, 2005; 51(8): 1331 - 1332. [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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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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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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W. E. Highsmith Jr SNPs for Sale. Cheap! Clin. Chem., August 1, 2004; 50(8): 1296 - 1298. [Full Text] [PDF]
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