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Distinguishing Different DNA Heterozygotes by High-Resolution Melting

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;

^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

High-resolution melting was recently introduced as a technique to genotype single-nucleotide polymorphisms (SNPs) within small amplicons (1). This closed-tube 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)(5)(6). The process is made possible by heteroduplex-detecting DNA dyes that can be used at saturating concentrations without inhibiting PCR (7). Wild-type and homozygous mutant samples are distinguished by melting temperature (T_m) shifts. Heterozygous samples are best distinguished from homozygotes, not by T_m, 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, nearest-neighbor 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 (HybProbe^TM) 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 (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 T_m become obvious.

Predicted duplex T_ms 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 T_m) or influence of LCGreen PLUS (tends to increase T_m) were used. To maximize differences in T_m, amplicon lengths were kept short (1), although SNP typing has been demonstrated in products more than 500 bp long (7). Calculated T_m differences between homozygous wild-type 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 T_ms. These unique melting curve shapes are best seen with normalized, temperature-shifted 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) 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).

View larger version (19K): [in this window] [in a new window]   Figure 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.

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 T_ms 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)(12)(13)(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 T_m to distinguish variants (14).

High-resolution melting analysis introduces the use of melting curve shape, as well as T_m, to distinguish different variants. Because the composite melting curves of heterozygotes include 2 homoduplexes and 2 heteroduplexes, the meaning of T_m is ambiguous and less useful as a metric than is curve shape. Is the T_m 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)? These are not identical because such curves are skewed at low temperatures from heteroduplex contributions. In either case, the T_m 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.

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This Article Extract Full Text (PDF) Data Supplements All Versions of this Article: clinchem.2005.051516v1 51/7/1295    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (32) Citing Articles via Google Scholar Google Scholar Articles by Graham, R. Articles by Wittwer, C. T. Search for Related Content PubMed PubMed Citation Articles by Graham, R. Articles by Wittwer, C. T. Related Collections Molecular Diagnostics and Genetics Hemostasis and Thrombosis Automation and Analytical Techniques