| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Molecular Diagnostics and Genetics |
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. 3 Department of Mathematics, University of Utah, Salt Lake City, UT.
aAddress correspondence to this author at: Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132. Fax 801-581-4517; e-mail carl.wittwer{at}path.utah.edu.
Background: High-resolution melting of PCR amplicons with the DNA dye LCGreenTM I was recently introduced as a homogeneous, closed-tube method of genotyping that does not require probes or real-time PCR. We adapted this system to genotype single-nucleotide polymorphisms (SNPs) after rapid-cycle PCR (12 min) of small amplicons (
50 bp).
Methods: Engineered plasmids were used to study all possible SNP base changes. In addition, clinical protocols for factor V (Leiden) 1691G>A, prothrombin 20210G>A, methylenetetrahydrofolate reductase (MTHFR) 1298A>C, hemochromatosis (HFE) 187C>G, and ß-globin (hemoglobin S) 17A>T were developed. LCGreen I was included in the reaction mixture before PCR, and high-resolution melting was obtained within 2 min after amplification.
Results: In all cases, heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves. Approximately 84% of human SNPs involve a base exchange between A::T and G::C base pairs, and the homozygotes are easily genotyped by melting temperatures (Tms) that differ by 0.8–1.4 °C. However, in 
16% of SNPs, the bases only switch strands and preserve the base pair, producing very small Tm differences between homozygotes (<0.4 °C). Although most of these cases can be genotyped by Tm, one-fourth (4% of total SNPs) show nearest-neighbor symmetry, and, as predicted, the homozygotes cannot be resolved from each other. In these cases, adding 15% of a known homozygous genotype to unknown samples allows melting curve separation of all three genotypes. This approach was used for the HFE 187C>G protocol, but, as predicted from the sequence changes, was not needed for the other four clinical protocols.
Conclusions: SNP genotyping by high-resolution melting analysis is simple, rapid, and inexpensive, requiring only PCR, a DNA dye, and melting instrumentation. The method is closed-tube, performed without probes or real-time PCR, and can be completed in less than 2 min after completion of PCR.
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 L. S. Kristensen and A. Dobrovic Direct Genotyping of Single Nucleotide Polymorphisms in Methyl Metabolism Genes Using Probe-Free High-Resolution Melting Analysis Cancer Epidemiol. Biomarkers Prev., May 1, 2008; 17(5): 1240 - 1247. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
J. Li, R. Berbeco, R. J. Distel, P. A. Janne, L. Wang, and G. M. Makrigiorgos s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis Nucleic Acids Res., June 9, 2007; 35(12): e84 - e84. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Palais Quantitative Heteroduplex Analysis Clin. Chem., June 1, 2007; 53(6): 1001 - 1003. [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
C. L.H. Lo, S. P. Yip, P. K.C. Cheng, T. S.S. To, W. W.L. Lim, and P. H.M. Leung One-Step Rapid Reverse Transcription-PCR Assay for Detecting and Typing Dengue Viruses with GC Tail and Induced Fluorescence Resonance Energy Transfer Techniques for Melting Temperature and Color Multiplexing Clin. Chem., April 1, 2007; 53(4): 594 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Glynou, P. Kastanis, S. Boukouvala, V. Tsaoussis, P. C. Ioannou, T. K. Christopoulos, J. Traeger-Synodinos, and E. Kanavakis High-Throughput Microtiter Well-Based Chemiluminometric Genotyping of 15 HBB Gene Mutations in a Dry-Reagent Format Clin. Chem., March 1, 2007; 53(3): 384 - 391. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
O. Alsmadi, F. Alkayal, M. Al-Sayed, M. S. Rashed, F. Imtiaz, and B. F. Meyer LCGreen I-Based Real-Time PCR Assays for Detecting Common ASL and HMGCL Variants. Clin. Chem., July 1, 2006; 52(7): 1439 - 1440. [Full Text] [PDF]
|
|
|
|
|
|
M. Erali, J. I. Pounder, G. L. Woods, C. A. Petti, and C. T. Wittwer Multiplex single-color PCR with amplicon melting analysis for identification of Aspergillus species. Clin. Chem., July 1, 2006; 52(7): 1443 - 1445. [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
W. Yang, T. Yaoi, S. Huang, Q. Yang, S. Hatcher, H. Seet, and J. P. Gregg Detecting the C282Y and H63D Mutations of the HFE Gene by Holliday Junction-Based Allele-Specific Genotyping Methods Clin. Chem., January 1, 2005; 51(1): 210 - 213. [Full Text] [PDF]
|
|
|
|
|
|
R. Rej Clinical Chemistry through Clinical Chemistry: A Journal Timeline Clin. Chem., December 1, 2004; 50(12): 2415 - 2458. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
