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Accessing genetic variation: genotyping single nucleotide polymorphisms [Review]. Nat Rev Genet 2001;2:930 – 42. 16. Bradford LD. CYP2D6 allele frequency in European Caucasians, Asians, Africans and their descendants [Review]. Pharmacogenomics 2002;3:229 – 43. 17. Lundqvist E, Johansson I, Ingelman-Sundberg M. Genetic mechanisms for duplication and multiduplication of the human CYP2D6 gene and methods for detection of duplicated CYP2D6 genes. Gene 1999;226:327–38. 18. Lovlie R, Daly AK, Molven A, Idle JR, Steen VM. Ultrarapid metabolizers of debrisoquine: characterization and PCR-based detection of alleles with duplication of the CYP2D6 gene. FEBS Lett 1996;392:30 – 4. 19. Steen VM, Andreassen OA, Daly AK, Tefre T, Borresen AL, Idle JR, et al. Detection of the poor metabolizer-associated CYP2D6(D) gene deletion allele by long-PCR technology. Pharmacogenetics 1995;5:215–23. 20. Cann HM, de Toma C, Cazes L, Legrand MF, Morel V, Piouffre L, et al. A human genome diversity cell line panel. Science 2002;296:261–2. 21. Fuselli S, Dupanloup I, Frigato E, Cruciani F, Scozzari R, Moral P, et al. Molecular diversity at the CYP2D6 locus in the Mediterranean region. Eur J Hum Genet 2004;12:916 –24. 22. Levo A, Koski A, Ojanperä I, Vuori E, Sajantila A. Post-mortem SNP analysis of CYP2D6 gene reveals correlation between genotype and opioid drug (tramadol) metabolite ratios in blood. Forensic Sci Int 2003;135:9 –15. 23. Johansson I, Lundqvist E, Dahl ML, Ingelman-Sundberg M. PCR-based genotyping for duplicated and deleted CYP2D6 genes. Pharmacogenetics 1996;6:351–5. 24. Bender K. SNaPshot for pharmacogenetics by minisequencing. Methods Mol Biol 2004;297:243–52. 25. Knaapen AM, Ketelslegers HB, Gottschalk RW, Janssen RG, Paulussen AD, Smeets HJ, et al. Simultaneous genotyping of nine polymorphisms in xenobiotic-metabolizing enzymes by multiplex PCR amplification and single base extension [Technical Brief]. Clin Chem 2004;50:1664 – 8. 26. Schultz J. FDA guidelines on race and ethnicity: obstacle or remedy? J Natl Cancer Inst 2003;95:425– 6. 27. Raimundo S, Toscano C, Klein K, Fischer J, Griese EU, Eichelbaum M, et al. A novel intronic mutation, 2988G⬎A, with high predictivity for impaired function of cytochrome P450 2D6 in white subjects. Clin Pharmacol Ther 2004;76:128 –38. Previously published online at DOI: 10.1373/clinchem.2004.046466 Distinguishing Different DNA Heterozygotes by HighResolution Melting, Robert Graham,1 Michael Liew,2 Cindy Meadows,2 Elaine Lyon,2 and Carl T. Wittwer1,2* (1 Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT; 2 Institute for Clinical and Experimental Pathology, ARUP, Salt Lake City UT; * address 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@ path.utah.edu) High-resolution melting was recently introduced as a technique to genotype single-nucleotide polymorphisms (SNPs) within small amplicons (1 ). This closed-tube 1295 method (including rapid-cycle PCR) can be completed in ⬍15 min and does not require real-time PCR instruments (2 ), allele-specific PCR (3 ), or fluorescently labeled oligonucleotides (4 – 6 ). The process is made possible by heteroduplex-detecting DNA dyes that can be used at saturating concentrations without inhibiting PCR (7 ). Wildtype and homozygous mutant samples are distinguished by melting temperature (Tm) shifts. Heterozygous samples are best distinguished from homozygotes, not by Tm, but by altered curve shape. Heterozygous samples produce heteroduplexes that melt at lower temperatures than homoduplexes. Melting curves of amplified heterozygotes include 2 homoduplexes and 2 heteroduplexes, giving a skewed composite melting curve easily distinguished from the curves for homozygotes by curve shape (1 ). However, it was not clear whether different heterozygotes within the same amplicon could be distinguished from each other based on curve shape differences. Four different classes of SNPs have been defined based on the homo- and heteroduplexes that are produced after amplification (1 ). Because the heteroduplex mismatches of SNPs in different classes are different, their melting curves should be distinguishable if the resolution of the instrumentation is sufficient. It is also possible that different heterozygotes in the same class may be distinguishable. Although the mismatches are the same, nearestneighbor stability parameters depend on the bases adjacent to the mismatches; therefore, the predicted stabilities are often different. We selected DNA samples retrospectively from those submitted to ARUP Laboratories for standard clinical genotyping of HFE, factor V Leiden, and factor II polymorphisms. DNA was extracted from blood samples by use of the MagNa Pure instrument (Roche) and genotyped by use of adjacent hybridization probe (HybProbeTM) methods (4, 5 ) on a LightCycler®. One advantage of probe melting analysis for genotyping is that sequence alterations other than those expected can often be identified under the probes (8 ). During routine clinical analysis at ARUP, when aberrant melting peaks are observed at unexpected temperatures, samples are sequenced to identify the sequence alteration. Several heterozygous sequence variants near the expected mutations were thus identified. Three factor II heterozygotes (all SNPs), 4 factor V heterozygotes (2 SNPs, 1 single-base deletion, and 1 compound heterozygote), and 6 HFE heterozygotes (4 SNPs and 2 compound heterozygotes) were available for comparison. After selection and deidentification of samples, DNA concentrations were checked on an ND-1000 (NanoDrop Technologies, Inc.). When available, 3 individuals of each heterozygous genotype were processed. Previously described primers were used (1 ) unless it was necessary to redesign them to flank all sequence variants. SNPWizard (http://DNAWizards.path.utah.edu) was used for primer design. Primers were synthesized by Integrated DNA Technologies and used without further purification. PCR was performed as described previously 1296 Technical Briefs (1 ), except that 1X LCGreen ® PLUS (Idaho Technology) replaced LCGreen I. LCGreen PLUS is brighter than LCGreen I and can be used on a wider variety of melting instruments. The amplicon sequences, primer regions, and specific PCR conditions used for amplification are listed in Table 1 of the Data Supplement that accompanies the online version of this Technical Brief at http:// www.clinchem.org/content/vol51/issue7/. After PCR, capillaries were heated to 90 °C at 20 °C/s in the LightCycler and then cooled to 40 °C at 20 °C/s to favor heteroduplex formation (9 ). Samples were removed from the LightCycler and analyzed on a high-resolution melting instrument (HR-1; Idaho Technology). Each capillary was inserted into the instrument at 59 °C, the temperature was increased at 0.3 °C/s, and fluorescence was acquired from 65 to 95 °C, which required only 1–2 min. Heteroduplex detection in small amplicons is favored by rapid cooling before melting, rapid heating during melting, and low Mg2⫹ concentrations (9 ). Melting curves were analyzed by custom software with normalized, temperature-shifted curves displayed as difference plots (1, 7 ). Difference plots display the difference between each sample and a “standard” (often the average of multiple wild-type curves) and should not be confused with derivative plots (⫺dF/dT), which are often used to analyze low-resolution melting data. Difference plots are a convenient way of viewing high-resolution melting data because slight differences in curve shape and Tm become obvious. Predicted duplex Tms were calculated with previously described nearest-neighbor thermodynamic models (1, 10 ) as implemented in MeltingWizard (available at http://DNAWizards.path.utah.edu). No corrections for dUTP incorporation instead of dTTP (tends to lower Tm) or influence of LCGreen PLUS (tends to increase Tm) were used. To maximize differences in Tm, amplicon lengths were kept short (1 ), although SNP typing has been demonstrated in products more than 500 bp long (7 ). Calculated Tm differences between homozygous wildtype and homozygous mutant amplicons varied from 0.0 to 1.3 °C for the different SNPs (Table 2 of the online Data Supplement). For single SNPs, heteroduplex destabilization varied from 1.1 to 3.0 °C compared with the most stable homoduplex, or 0.7–2.2 °C compared with the least stable homoduplex. For compound heterozygotes, the respective destabilizations were 2.8 – 4.0 °C and 2.0 –3.5 °C. High-resolution melting techniques are necessary to differentiate different heterozygotes within the same amplicon. Melting curves generated on the LightCycler did not reliably separate heterozygotes (data not shown), in contrast to high-resolution HR-1 analysis. As seen in Fig. 1, each heterozygote traces a unique melting curve path according to its 4 duplex Tms. These unique melting curve shapes are best seen with normalized, temperatureshifted data, shown in the form of difference plots. In Fig. 1A, factor II and 2 rare heterozygotes (all class 1 SNPs) are clearly separated from the wild type and from each other. In Fig. 1B, factor V Leiden and 3 rare heterozygotes (one SNP, one 1-bp deletion, and one compound heterozygote) Fig. 1. Melting analysis of several heterozygous sequence variants near common mutations in factor II (20210G⬎A), factor V (Lieden; 1691G⬎A), and HFE (187C⬎G). The melting curves were normalized and temperature-shifted, and the difference between each sample and the average of the wild types is displayed at each temperature (1, 7 ). Such “difference” plots should not be confused with the commonly used derivative plots. Each mutation is indicated by its location (20210) followed by the base pair change (G⬎A). All samples are heterozygous or compound heterozygous, unless followed by a WT, which signifies the wild type. Three individuals of each genotype are shown, except for factor II 20218G⬎A and factor V compound heterozygote 1696A⬎G/1690G⬎A, which are in duplicate, and the HFE compound heterozygote 187C⬎G/189T⬎C, for which only 1 sample was available. Clinical Chemistry 51, No. 7, 2005 also followed distinct paths. As expected, single SNPs and the 1-bp deletion (single unpaired loci) were more similar to each other than the compound heterozygote (2 unpaired loci). In Fig. 1C, all heterozygotes tested were clearly separated from each other and the wild type, including the targeted HFE mutation, 3 rare SNPs, and 2 rare compound heterozygotes. In all cases studied, different heterozygotes could be distinguished, including those within the same SNP class (1 ). At the factor V and HFE loci, compound heterozygotes were easily distinguished from “single” heterozygotes, as might be expected because of 2, rather than 1, region of destabilization. It is interesting that the predicted Tms of the homoduplexes and/or heteroduplexes are different depending on the cis/trans orientation of the sequence variants (Table 2 of the online Data Supplement). This suggests that melting analysis may allow haplotyping, at least when the variations to be haplotyped are in the same melting domain. The presence of unexpected polymorphisms close to a SNP of interest may or may not interfere with genotyping, depending on the analysis method (10 ). All PCR-based methods can be compromised if polymorphisms occur under the primers and lead to undesired allele-specific PCR. The same concern applies to internal primers used for sequencing or extension reactions. These caveats aside, some PCR-based methods, including sequencing, will identify and distinguish polymorphisms that are missed by restriction fragment length polymorphism assays and isothermal probe-based assays. Hybridization probe assays that interrogate over a range of temperatures (melting assays) often detect the presence of unexpected polymorphisms but may require further studies to identify them. In our study, high-resolution melting of small amplicons distinguished all heterozygotes studied. This included 21 pairwise comparisons, suggesting that most randomly selected heterozygotes within small amplicons can be distinguished. The power of melting analysis to distinguish multiple sequence variants has been controversial (11–14 ). When wild-type probes are used on standard low-resolution systems, 19%–52% of possible SNP heterozygotes may be confused with the expected heterozygous variant (11 ). Although better temperature resolution has been reported on these instruments (12, 13 ), it is clear that rare sequence variants under a probe may not be distinguished from the common targeted variant. Similar concerns for genotyping by amplicon melting temperature have been described and focus on the limitations of Tm to distinguish variants (14 ). High-resolution melting analysis introduces the use of melting curve shape, as well as Tm, to distinguish different variants. Because the composite melting curves of heterozygotes include 2 homoduplexes and 2 heteroduplexes, the meaning of Tm is ambiguous and less useful as a metric than is curve shape. Is the Tm of a composite melting curve the temperature at which one-half of the duplexes have melted (conventional definition), or is it the peak of the derivative plot (common derivation)? 1297 These are not identical because such curves are skewed at low temperatures from heteroduplex contributions. In either case, the Tm is only one point on the melting curve. Use of the complete melting curve, conveniently displayed as difference plots, allows differentiation of most heterozygotes (21 of 21 random pairwise comparisons in this study). High-resolution melting analysis is convenient because no processing or separation steps are required (7 ). In cases in which the complexity of the target exceeds the genotyping capabilities of amplicon melting, unlabeled oligonucleotide probes can be added (15 ). In addition to genotyping, high-resolution melting analysis is an accurate mutation scanning tool (16 ) that has been applied to the medium chain acyl-CoA dehydrogenase (17 ), c-kit (18 ), and SLC22A5 (19 ) genes, as well as HLA matching (20 ). The sensitivity and specificity are greater than denaturing HPLC (21 ), and no electrophoresis is required, as in temperature gradient (22 ), denaturing gradient (23 ), or conformation-sensitive (24 ) gel electrophoresis. References 1. Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, et al. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem 2004;50:1156 – 64. 2. Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA, Balis UJ. 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Detection and identification of base alterations within the region of factor V Leiden by fluorescent melting curves. Mol Diagn 1998;3:203–10. 14. von Ahsen N, Oellerich M, Schutz E. Limitations of genotyping based on amplicon melting temperature. Clin Chem 2001;47:1331–2. 15. Zhou L, Myers AN, Vandersteen JG, Wang L, Wittwer CT. Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye. Clin Chem 2004;50:1328 –35. 16. Reed GH, Wittwer CT. Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem 2004; 50:1748 –54. 17. McKinney JT, Longo N, Hahn SH, Matern D, Rinaldo P, Strauss AW, et al. Rapid, comprehensive screening of the human medium chain acyl-CoA dehydrogenase gene. Mol Genet Metab 2004;82:112–20. 18. Willmore C, Holden JA, Zhou L, Tripp S, Wittwer CT, Layfield LJ. Detection of 1298 Technical Briefs c-kit-activating mutations in gastrointestinal stromal tumors by high-resolution amplicon melting analysis. Am J Clin Pathol 2004;122:206 –16. 19. Dobrowolski SF, McKinney JT, Amat di San Filippo C, Giak Sim K, Wilcken B, Longo N. Validation of dye-binding/high-resolution thermal denaturation for the identification of mutations in the SLC22A5 gene. Hum Mutat 2005;25: 306 –13. 20. Zhou L, Vandersteen J, Wang L, Fuller T, Taylor M, Palais B, et al. High-resolution DNA melting curve analysis to establish HLA genotypic identity. Tissue Antigens 2004;64:156 – 64. 21. Chou LS, Lyon E, Wittwer CT. A comparison of high-resolution melting analysis to denaturing high performance liquid chromatography for mutation scanning: cystic fibrosis transmembrane conductance regulator gene as a model. Am J Clin Pathol 2005;in press. 22. Wartell RM, Hosseini S, Powell S, Zhu J. Detecting single base substitutions, mismatches and bulges in DNA by temperature gradient gel electrophoresis and related methods. J Chromatogr A 1998;806:169 – 85. 23. Abrams ES, Murdaugh SE, Lerman LS. Comprehensive detection of single base changes in human genomic DNA using denaturing gradient gel electrophoresis and a GC clamp. Genomics 1990;7:463–75. 24. Nataraj AJ, Olivos-Glander I, Kusukawa N, Highsmith WE Jr. Single-strand conformation polymorphism and heteroduplex analysis for gel-based mutation detection. Electrophoresis 1999;20:1177– 85. Previously published online at DOI: 10.1373/clinchem.2005.051516