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Automation and Analytical Techniques |
1 Institute for Clinical and Experimental Pathology, ARUP, Salt Lake City, UT.
2 Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT.
aAddress correspondence to this author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108. Fax 801-584-5114; e-mail mark.herrmann{at}aruplab.com.
Background: DNA melting analysis for genotyping and mutation scanning of PCR products by use of high-resolution instruments with special "saturation" dyes has recently been reported. The comparative performance of other instruments and dyes has not been evaluated.
Methods: A 110-bp fragment of the ß-globin gene including the sickle cell anemia locus (A17T) was amplified by PCR in the presence of either the saturating DNA dye, LCGreen Plus, or SYBR Green I. Amplicons of 3 different genotypes (wild-type, heterozygous, and homozygous mutants) were melted on 9 different instruments (ABI 7000 and 7900HT, Bio-Rad iCycler, Cepheid SmartCycler, Corbett Rotor-Gene 3000, Idaho Technology HR-1 and LightScanner, and the Roche LightCycler 1.2 and LightCycler 2.0) at a rate of 0.1 °C/s or as recommended by the manufacturer. The ability of each instrument/dye combination to genotype by melting temperature (Tm) and to scan for heterozygotes by curve shape was evaluated.
Results: Resolution varied greatly among instruments with a 15-fold difference in Tm SD (0.018 to 0.274 °C) and a 19-fold (LCGreen Plus) or 33-fold (SYBR Green I) difference in the signal-to-noise ratio. These factors limit the ability of most instruments to accurately genotype single-nucleotide polymorphisms by amplicon melting. Plate instruments (96-well) showed the greatest variance with spatial differences across the plates. Either SYBR Green I or LCGreen Plus could be used for genotyping by Tm, but only LCGreen Plus was useful for heterozygote scanning. However, LCGreen Plus could not be used on instruments with an argon laser because of spectral mismatch. All instruments compatible with LCGreen Plus were able to detect heterozygotes by altered melting curve shape. However, instruments specifically designed for high-resolution melting displayed the least variation, suggesting better scanning sensitivity and specificity.
Conclusion: Different instruments and dyes vary widely in their ability to genotype homozygous variants and scan for heterozygotes by whole-amplicon melting analysis.
The following articles in journals at HighWire Press have cited this article:
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  O.P. Hurtado-Gonzales, L.M. Aragon-Caballero, J.G. Flores-Torres, W. M. i. t Veld, and K.H. Lamour Molecular comparison of natural hybrids of Phytophthora nicotianae and P. cactorum infecting loquat trees in Peru and Taiwan Mycologia, July 1, 2009; 101(4): 496 - 502. [Abstract] [Full Text] [PDF]
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 A. T. Pietzka, A. Indra, A. Stoger, J. Zeinzinger, M. Konrad, P. Hasenberger, F. Allerberger, and W. Ruppitsch Rapid identification of multidrug-resistant Mycobacterium tuberculosis isolates by rpoB gene scanning using high-resolution melting curve PCR analysis J. Antimicrob. Chemother., June 1, 2009; 63(6): 1121 - 1127. [Abstract] [Full Text] [PDF]
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 H. Ye, M. C. Huang, M.-H. Li, and J. Y. Ying Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis Nucleic Acids Res., April 1, 2009; 37(7): e51 - e51. [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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 P. A. Steer, N. C. Kirkpatrick, D. O'Rourke, and A. H. Noormohammadi Classification of Fowl Adenovirus Serotypes by Use of High-Resolution Melting-Curve Analysis of the Hexon Gene Region J. Clin. Microbiol., February 1, 2009; 47(2): 311 - 321. [Abstract] [Full Text] [PDF]
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R. S. Donahoo and K. H. Lamour Interspecific hybridization and apomixis between Phytophthora capsici and Phytophthora tropicalis. Mycologia, November 1, 2008; 100(6): 911 - 920. [Abstract] [Full Text] [PDF]
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 B. J. Wolff, W. L. Thacker, S. B. Schwartz, and J. M. Winchell Detection of Macrolide Resistance in Mycoplasma pneumoniae by Real-Time PCR and High-Resolution Melt Analysis Antimicrob. Agents Chemother., October 1, 2008; 52(10): 3542 - 3549. [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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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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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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 D. Szumska, G. Pieles, R. Essalmani, M. Bilski, D. Mesnard, K. Kaur, A. Franklyn, K. El Omari, J. Jefferis, J. Bentham, et al. VACTERL/caudal regression/Currarino syndrome-like malformations in mice with mutation in the proprotein convertase Pcsk5 Genes & Dev., June 1, 2008; 22(11): 1465 - 1477. [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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 K. E Liyanage, A. J Hooper, J. C Defesche, J. R Burnett, and F. M van Bockxmeer High-resolution melting analysis for detection of familial ligand-defective apolipoprotein B-100 mutations Ann Clin Biochem, March 1, 2008; 45(2): 170 - 176. [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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C. Dahl and P. Guldberg High-Resolution Melting for Accurate Assessment of DNA Methylation Clin. Chem., November 1, 2007; 53(11): 1877 - 1878. [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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H. Gudnason, M. Dufva, D.D. Bang, and A. Wolff Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature Nucleic Acids Res., October 8, 2007; 35(19): e127 - e127. [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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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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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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 E. P. Price, H. Smith, F. Huygens, and P. M. Giffard High-Resolution DNA Melt Curve Analysis of the Clustered, Regularly Interspaced Short-Palindromic-Repeat Locus of Campylobacter jejuni Appl. Envir. Microbiol., May 15, 2007; 73(10): 3431 - 3436. [Abstract] [Full Text] [PDF]
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M. L. Kennerson, T. Warburton, E. Nelis, M. Brewer, P. Polly, P. De Jonghe, V. Timmerman, and G. A. Nicholson Mutation Scanning the GJB1 Gene with High-Resolution Melting Analysis: Implications for Mutation Scanning of Genes for Charcot-Marie-Tooth Disease Clin. Chem., February 1, 2007; 53(2): 349 - 352. [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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C. Nellaker, U. Wallgren, and H. Karlsson Molecular Beacon-Based Temperature Control and Automated Analyses for Improved Resolution of Melting Temperature Analysis Using SYBR I Green Chemistry Clin. Chem., January 1, 2007; 53(1): 98 - 103. [Abstract] [Full Text] [PDF]
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