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1 Department of Pathology, University of Utah Medical School, Salt Lake City, UT 84132.
aAuthor for correspondence. Fax 801-581-4517; e-mail carl.wittwer{at}path.utah.edu.
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Abstract |
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Methods: We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), ß-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR.
Results: The six common ß-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve acquisition required only 1–2 min, amplification and analysis were achieved in 10–20 min with rapid cycling conditions.
Conclusions: High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR for amplicon genotyping, and is a promising method for mutation screening.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two adjacent hybridization probes are most commonly used for melting curve analysis of small sequence variations, but a single hybridization probe can also been used (9). With only a single probe, the fluorescence change observed with hybridization may be small, first suggesting the need for high-resolution melting analysis.
If PCR is performed with a 5'-labeled primer, high-resolution melting analysis of the amplicon can distinguish different homozygotes and heterozygotes (10). The ability to genotype single-nucleotide polymorphisms within amplicons up to 304 bp in length suggests that scanning for unknown polymorphisms may also be successful. Disadvantages of the method include the need for a labeled oligonucleotide and a requirement that the sequence alterations must be within the melting domain that includes the labeled primer.
Limitations in the use of SYBR Green I for detecting small sequence variations are detailed elsewhere (10). Briefly, detection of heterozygotes by SYBR Green I has been possible only when extra steps are added between amplification and analysis, such as amplicon purification and addition of high amounts of dye (11) or urea (12). A completely closed-tube method using a generic dsDNA dye for effective genotyping and scanning has been elusive. After screening several dsDNA dyes, we found one dye that did detect heterozygotes and at the same time did not inhibit or adversely affect PCR. In this report, we demonstrate the usefulness of LCGreen in genotyping applications that involve high-resolution melting curve analysis of PCR amplicons. We also introduce melting curve subtraction (fluorescence difference) as a useful method of differentiating genotypes that allows better visual grouping of genotypes than the more common first-derivative method. The potential of using LCGreen for unknown variant detection (so-called mutation scanning) is also considered.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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melting curve acquisition
Melting analysis was performed on the LightCycler or on a high-resolution melting instrument (HR-1; Idaho Technology), as described previously for labeled primers (10). After amplification, the samples were heated momentarily in the LightCycler to 94 °C and cooled to 40 °C unless stated otherwise. The LightCycler capillary was then transferred to the high-resolution melting instrument and heated at 0.3 °C/s unless otherwise stated. The HR-1 is a single-sample instrument that surrounds one LightCycler capillary with an aluminum cylinder that is heated by a coil wound around the outside. Sample temperature and fluorescence signals are converted to 16-bit digital signals, ideally providing resolution down to 0.002 °C and 0.002% of normalized fluorescence. Approximately 50 data points are acquired for every 1 °C.
melting curve analysis
LightCycler and high-resolution melting data were analyzed with custom software written in LabView (National Instruments). Fluorescence intensity values were usually normalized between 0% and 100% by first defining linear baselines before and after the melting transition of each sample. Within each sample, the fluorescence of each acquisition was calculated as the percentage of fluorescence between the top and bottom baselines at each acquisition temperature.
In some cases after normalization, the temperature axis of each curve was adjusted to superimpose the curves over a certain fluorescence interval. One sample was chosen arbitrarily, and the points within the fluorescence interval were fit to a quadratic. For each remaining curve, the required temperature shift for translation of each point within the fluorescence interval onto the quadratic was calculated. Each curve was then translated by the average shift to allow superimposition of the curves within the selected fluorescence interval.
Different genotypes were most easily discerned by plotting the fluorescence difference between normalized (and optionally, temperature-shifted) melting curves. One melting curve was chosen as a reference, and the difference between each curve and the reference was plotted against temperature to give a "fluorescence difference" plot. The original reference curve became a horizontal line at zero, and different genotypes clustered along different paths for easy visual discrimination.
Derivative melting curve plots were calculated from the Savitsky–Golay polynomials at each point (17). Savitsky–Golay analysis used a second-degree polynomial and a data window including all points within a 1 °C interval.
melting curve genotyping of cystic fibrosis mutations with sybr green i or LCGREEN
A 41- to 44-bp fragment was amplified with the primers 5'-GGCACCATTAAAGAAAATAT-3' and 5'-TCATCATAGGAAACACCA-3' (GenBank accession no. M55115; positions 413–456R) in the presence of SYBR Green I or 10 µM LCGreen. The primers flanked the mutational hot spot containing the F508del, I507del, and F508C variants. PCR was performed through 40 cycles of 85 and 58 °C (0 s holds). A final melting cycle was performed on the LightCycler by heating to 95 °C, cooling to 55 °C, and collecting fluorescence continuously at a ramping rate of 0.2 °C/s. Five samples were monitored during melting curve acquisition.
melting curve analysis of multiple products with sybr green i or LCGREEN
A low-mass DNA ladder (Invitrogen), usually used as a length marker for gel electrophoresis, was melted in the presence of SYBR Green I or LCGreen. The DNA ladder (235 ng) was mixed with a 1:10 000 dilution of SYBR Green I or 4 µM LCGreen in the presence of 50 mM Tris (pH 8.3), 3 mM MgCl2, and 500 ng/µL bovine serum albumin. These dye concentrations each produced 9% of the maximum fluorescence observed when excess dye was present. Each sample was melted at 0.1 °C/s in the high-resolution melting instrument. Derivative plots were obtained as described above.
ß-globin mutations (HBC and HBS)
A 110-bp fragment of the ß-globin gene was amplified with primers 5'-ACACAACTGTGTTCACTAGC-3' and 5'-CAACTTCATCCACGTTCACC-3' (GenBank accession no. GI:455025; positions 62150–62259R). The single-nucleotide polymorphisms hemoglobin (Hb)C (G16A) and HbS (A17T) are near the middle of this amplicon. DNA was extracted from dried blood spots of two different individuals with each of the common genotypes. The genotypes included three homozygous (AA, SS, and CC) and three heterozygous (AS, AC, and SC) types. Each reaction contained 10 µM LCGreen. After an initial denaturation for 5 s at 95 °C, the samples were cycled 35 times with the following protocol: 94 °C with no hold, 50 °C with a 2-s hold, and a 1 °C/s ramp to 72 °C with a 2-s hold. After PCR amplification, denaturation, and cooling, fluorescence was acquired while heating from 70 to 93 °C at a rate of 0.3 °C/s with the high-resolution melting instrument.
htr2a single-nucleotide polymorphism
A 544-bp genomic fragment was amplified from the human 5-hydroxytryptamine receptor 2A (HTR2A) gene (GenBank accession no. NM_000621.1) with primers 5'-CCAGCTCCGGGAGA-3' and 5'-CATACAGGATGGTTAACATGG-3' (positions 14–557R). We used 1 µM LCGreen and 2 mM MgCl2. PCR cycling conditions were as follows: 40 cycles of 92 °C for 0 s, 60 °C for 2 s, and 74 °C for 25 s. Duplicates of one patient sample of each genotype (CC, TC, and TT) were amplified and analyzed. After PCR amplification, denaturation, and cooling, fluorescence was acquired while heating from 70 to 93 °C at a rate of 0.3 °C/s in the high-resolution melting instrument. A melting temperature (Tm) map of the homozygous wild-type sequence was obtained using Poland (18) and Fixman–Freire (19) algorithms with the MELT94 software (available athttp://web.mit.edu/osp/www/melt.html).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Two homozygous and three heterozygous genotypes were analyzed.
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Heteroduplexes were not detected with ethidium bromide, SYBR Green I, SYBR Gold, Pico Green, TOTO-1, and YOYO-1 dyes. The derivative melting curves for SYBR Green I are shown in Fig. 1B
. All genotypes showed symmetric derivative curves with Tms within 1 °C of each other. The Tm of the F508C heterozygote was slightly higher than that of the wild type, reflecting the greater stability of a GC vs an AT base pair (13). The three genotypes containing heterozygous or homozygous deletions melted at a slightly lower temperature than the wild type and were difficult to distinguish from each another.
In contrast, amplification and melting of heterozygotes in the presence of a newly available dye, LCGreen, produced two clearly defined peaks (Figs. 1C
). Similar to the results obtained with labeled primers (10), the lower-temperature peak was always smaller than the higher-temperature peak, indicating the melting transition of a heteroduplex product. As might be expected, the heterozygotes with 3 bp deleted (F508del and I507del) produced heteroduplex products that were more destabilized than heteroduplex products from a single base change (F508C). With LCGreen, the major melting transition was spread over 2 °C, approximately twice the range observed with SYBR Green I. This greater range, in addition to the presence of heteroduplex peaks, allowed complete differentiation of all five genotypes. When the concentration of LCGreen was varied from 1 to 60 µM, there was little difference in the percentage area of the heterozygous peak (data not shown). The mean (SD) area for the heterozygote was 12.7 (2.7)% (n = 7 different concentrations).
dye redistribution during melting
One reason that LCGreen identifies low-melting transitions better than SYBR Green I is illustrated in Fig. 2
. When several DNA fragments of increasing thermal stability are present, the low-temperature peaks are very small with SYBR Green I compared with LCGreen. During melting, SYBR Green I may be released from low-temperature duplexes only to attach to duplexes that melt at higher temperatures. This causes each successive peak to be higher than the last. In contrast, LCGreen shows clear peaks for all duplexes, and the peak height does not always increase with Tm. Compared with SYBR Green I, LCGreen does not appear to redistribute during melting.
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The area of each peak in Fig. 2
was determined and divided by the known amount of each DNA species, providing an assessment of the relative sensitivity of each dye for each band (data not shown). LCGreen displayed a tendency for higher sensitivity with low-temperature melting species. In contrast, SYBR Green I strongly favored species that melted at higher temperatures.
analysis methods for heteroduplex and homoduplex differentiation
Melting curve analysis of six common genotypes of ß-globin in a 110-bp amplicon is detailed in Fig. 3
. The original high-resolution data are shown in Fig. 3A
. Note that the magnitude of the fluorescence was variable between different samples because of differences in the starting template and/or capillary optics. It is difficult to see any consistent difference between genotypes. The derivative plot of this data also did not clearly distinguish genotypes, although the presence of heterozygotes could be identified (data not shown).
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The magnitude differences between samples in the original data could be normalized. Two linear regions were selected, one before and one after the major transition. These regions defined two lines for each curve, an upper, 100% fluorescence line and a lower, 0% fluorescence line. The percentage of fluorescence within the transition (between the two regions) was calculated at each temperature as the distance to the experimental data compared with the distance between the extrapolated upper and lower lines. The normalized result for the ß-globin data is shown in Fig. 3B
. Each genotype now grouped together, most apparent between 84 and 85 °C. Different homozygotes had similar shapes but different Tms, differing by 0.2–0.4 °C. Although there was some variation within each genotype (0.1–0.2 °C), all homozygotes were clearly differentiated when the entire curve was considered. Heterozygotes have a different shape than homozygotes in the low melting region of the transition.
When the purpose of analysis is to scan for heterozygotes, temperature offsets between samples can be eliminated by shifting the temperature axis of each curve so that the curves are superimposed over a given fluorescence interval. Amplification of a heterozygote produces both low-temperature heteroduplex transitions and high-temperature homoduplex transitions. When the curves were shifted to superimpose their high-temperature, homoduplex regions (low percentage of fluorescence), heteroduplexes could be identified by their early decrease in fluorescence at lower temperatures, as shown in Fig. 3C
. However, this temperature shift would reduce or possibly eliminate any difference between homozygotes. That is, temperature-shifting alters the apparent Tm (by up to 0.4 °C in Fig. 3
) and should not be used for calculating Tms.
Different genotypes were most easily distinguished by plotting the fluorescence difference between normalized (and optionally, temperature-shifted) melting curves. A reference genotype was first selected (AA in Fig. 3
). The difference between all other genotype curves and the reference was then plotted against temperature, as shown in Fig. 3D
. The reference sample (subtracted from itself) became zero across all temperatures. Other genotypes traced unique paths that could be identified by visual pattern matching. Each individual (two for each genotype) was run in duplicate to get some indication of the variation between individuals and between genotypes as well as the experimental variation from amplification and melting. Because the duplicates of each individual did not appear to cluster, individual variation does not appear to interfere with the difference between genotypes.
single-nucleotide polymorphism typing in long pcr amplicons with multiple domains
High-resolution melting analysis of a single-nucleotide polymorphism within a 544-bp fragment of the human HTR2A gene is shown in Fig. 4
. Duplicate samples of each genotype (CC, TC, and TT) were amplified and analyzed. The data were normalized and temperature-shifted as described for ß-globin genotyping with curve superimposition between 10% and 20% fluorescence. The inset in Fig. 4
shows a predicted melting map of the homoduplex and the position of the polymorphism in the lower melting domain. The experimental data also show two apparent melting domains. All genotypes were similar in the higher melting domain. Genotypes differed in the lower melting domain, and the heterozygote pattern resembled that observed with labeled primers (10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this report, we describe a closed-tube method for genotyping entire amplicons that uses the dsDNA dye LCGreen in combination with high-resolution melting curve analysis. In a short cystic fibrosis model system (10), most of the dsDNA dyes commonly used in real-time PCR did not differentiate heterozygotes from homozygotes. The dyes SYBR Green I (Fig. 1B
), ethidium bromide, SYBR Gold, Pico Green, TOTO-1, and YOYO-1 did not reveal heteroduplexes when tested at noninhibitory concentrations.
LCGreen is a dsDNA dye that detects heteroduplexes (Fig. 1C
), accurately genotypes amplicons (Figs. 3
and 4
), and could be useful for closed-tube mutation scanning. Not only are heteroduplexes detected, the major melting transitions observed with LCGreen are expanded between genotypes compared with SYBR Green I (Fig. 1
). This expansion is particularly useful in differentiating different homozygotes. Concentrations between 1 and 10 µmol/L are most useful. At concentrations <1 µmol/L, the signal-to-noise ratio decreases, and at concentrations >10 µmol/L, the melting transition broadens and genotyping is not as reproducible (data not shown).
The reason that LCGreen detects heteroduplexes whereas SYBR Green I does not is not entirely clear. Saturation curves can be obtained by measuring the fluorescence of different dye dilutions with the same amount of DNA (data not shown). If 100 ng/10 µL is chosen as the amount of DNA present at the PCR plateau, LCGreen can be used at >90% saturation, whereas SYBR Green I completely inhibits PCR at >50% saturation. Dye redistribution during acquisition of the melting curve may be one reason that heteroduplexes are not observed with SYBR Green I (10). However, even when the dyes were used at the same low saturation concentration (Fig. 2
), melting results obtained with LCGreen were much more representative of the products present than results obtained with SYBR Green I, which is strongly biased against low-melting products.
Heteroduplexes resulting from the amplification of heterozygotes are easier to detect when the PCR product is short. With the 44-bp cystic fibrosis amplicon, the heteroduplex transition was clearly observed on derivative plots (Fig. 1
). When we (20) used engineered plasmid templates from a DNA mutation toolkit (Cambrex Bio Science Rockland, Inc.), the LightCycler could detect all possible heterozygotes with short amplicons (data not shown). However, as amplicon size increases, it becomes increasingly difficult to identify heterozygotes. The heteroduplex effects on melting curves are still present and could be used for genotyping and scanning if high-resolution methods are used. The requirements of such an approach include not only high-resolution instrumentation, but also adequate analysis techniques. For example, conventional derivative plots limit the display of high-resolution data because taking a derivative necessarily smoothes the data and the effect of heteroduplexes may be obscured in the rising shoulder of the major transition.
High-resolution techniques may also be needed to distinguish different homozygotes from each other. A similar situation occurs when the DNA analyzed is not diploid, such as with most genes in bacteria or viruses. For example, the Tm difference between some homoduplexes (single base change of A:T to T:A or G:C to C:G) challenges even high-resolution methods. One way to detect homozygous variants with nearly identical Tms is to mix the variants together. If the samples are different, heteroduplexes will be easily identified.
On first glance, original melting curve data do not appear to contain enough information for genotyping (Fig. 3A
). Conventional derivative plots are also not very informative (not shown). However, simple normalization of the fluorescence before and after the melting transitions allows visual clustering of different genotypes (Fig. 3B
). If the purpose of analysis is to scan for heterozygotes, temperature-shifting each sample by superimposing the normalized curves at low fluorescence (high temperature) is helpful. Heterozygotes are identifiable by an early decrease in fluorescence before the major melting transition (Fig. 3C
). Different heterozygotes trace different paths defined by the unique heteroduplexes and homoduplexes present. The differences between genotypes become even more apparent when displayed as fluorescence difference plots (Fig. 3D
). These difference plots should not be confused with conventional derivative plots. Surprisingly, different homozygotes appear distinguishable after normalization, temperature-shifting, and difference analysis. This implies that the shape of the melting curve, as well as the overall Tm, may vary among different homozygous genotypes. Although automatic clustering algorithms could be devised for genotyping, simple visual clustering on difference plots appears simple and accurate.
Although short amplicons produce greater genotyping differences, larger amplicons can also be genotyped. DNA melting domains are usually 
50 to 300 bp in length (21), and larger amplicons, e.g., 100–800 bp, may have multiple melting domains. A sequence alteration in one domain may not affect the melting of other domains. The 544-bp HTR2A amplicon contains two melting domains, both by calculation [Fig. 4
, inset, and Refs. (18)(19)] and experiment (Fig. 4
). The polymorphism is in the lower melting domain, and the melting curves of the different genotypes follow the expected patterns observed for single-nucleotide polymorphisms (10).
Because the melting of one domain appears to be independent of the melting of other domains, an invariant domain may be used as an internal control to adjust the temperature axis to correct instrument and/or sample run variation. Alternatively, temperature calibration of each curve could be provided by control DNA added to each sample before amplification or melting. Because multiple melting domains are present in larger amplicons, the variation in shape may occur in any portion of the curve, and it should be possible to identify the heterozygous domain. Because amplicon melting splits into domains, there may not be an upper limit on the amplicon size for genotyping and mutation scanning. Similar to mass spectrometry, amplicon melting analysis can become a more powerful tool with higher instrument resolution. Recently, we used this technique to distinguish six different genotypes within a 612-bp amplicon of the cystic fibrosis gene (data not shown). Our results suggest that when sufficient resolution is available, most sequence alterations of most amplicons can be genotyped in a single tube. However, potential confounding effects of DNA concentration and salt concentration (particularly Mg2+) should be considered and have been discussed previously (10). When labeled primers are used, heteroduplex formation is favored by lower Mg2+ concentrations. With LCGreen, the concentration of Mg2+ may also affect dye redistribution during melting.
Heteroduplex detection by high-resolution melting is particularly promising for identifying unknown sequence variants. However, the detection sensitivity remains to be established and will likely improve with advances in instrumentation and analysis methods. Compared with well-established techniques such as denaturing HPLC, high-resolution melting analysis for genotyping and mutation scanning has just begun.
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Acknowledgments |
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Footnotes |
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References |
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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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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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 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]
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H. Schonenbrucher, A. Abdulmawjood, K. Failing, and M. Bulte New Triplex Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Feces Appl. Envir. Microbiol., May 1, 2008; 74(9): 2751 - 2758. [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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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]
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A. J. Stephens, J. Inman-Bamber, P. M. Giffard, and F. Huygens High-Resolution Melting Analysis of the spa Repeat Region of Staphylococcus aureus Clin. Chem., February 1, 2008; 54(2): 432 - 436. [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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A. D. Laurie, M. P. Smith, and P. M. George Detection of Factor VIII Gene Mutations by High-Resolution Melting Analysis Clin. Chem., December 1, 2007; 53(12): 2211 - 2214. [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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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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T. Takano, Y. Ohe, K. Tsuta, T. Fukui, H. Sakamoto, T. Yoshida, U. Tateishi, H. Nokihara, N. Yamamoto, I. Sekine, et al. Epidermal Growth Factor Receptor Mutation Detection Using High-Resolution Melting Analysis Predicts Outcomes in Patients with Advanced Non Small Cell Lung Cancer Treated with Gefitinib Clin. Cancer Res., September 15, 2007; 13(18): 5385 - 5390. [Abstract] [Full Text] [PDF]
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 N. Jeffery, R. B. Gasser, P. A. Steer, and A. H. Noormohammadi Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region Microbiology, August 1, 2007; 153(8): 2679 - 2688. [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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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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D. Fortini, A. Ciammaruconi, R. De Santis, A. Fasanella, A. Battisti, R. D'Amelio, F. Lista, A. Cassone, and A. Carattoli Optimization of High-Resolution Melting Analysis for Low-Cost and Rapid Screening of Allelic Variants of Bacillus anthracis by Multiple-Locus Variable-Number Tandem Repeat Analysis Clin. Chem., July 1, 2007; 53(7): 1377 - 1380. [Abstract] [Full Text] [PDF]
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B. Palais Quantitative Heteroduplex Analysis Clin. Chem., June 1, 2007; 53(6): 1001 - 1003. [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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T. K. Wojdacz and A. Dobrovic Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation Nucleic Acids Res., March 19, 2007; 35(6): e41 - e41. [Abstract] [Full Text] [PDF]
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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]
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L. Stromqvist Meuzelaar, K. Hopkins, E. Liebana, and A. J. Brookes DNA Diagnostics by Surface-Bound Melt-Curve Reactions J. Mol. Diagn., February 1, 2007; 9(1): 30 - 41. [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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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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U. Reischl Melting of the Ribosomal RNA Gene Reveals Bacterial Species Identity: A Step toward a New Rapid Test in Clinical Microbiology. Clin. Chem., November 1, 2006; 52(11): 1985 - 1987. [Full Text] [PDF]
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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]
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J. Stenman, A. Paju, O. Rissanen, T. Tenkanen, C. Haglund, J. Rasanen, J. Salo, U.-H. Stenman, and A. Orpana Targeted Gene-Expression Analysis by Genome-Controlled Reverse Transcription-PCR Clin. Chem., November 1, 2006; 52(11): 1988 - 1996. [Abstract] [Full Text] [PDF]
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B. S. Robinson, P. T. Monis, and P. J. Dobson Rapid, Sensitive, and Discriminating Identification of Naegleria spp. by Real-Time PCR and Melting-Curve Analysis Appl. Envir. Microbiol., September 1, 2006; 72(9): 5857 - 5863. [Abstract] [Full Text] [PDF]
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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]
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M. Procter, L.-S. Chou, W. Tang, M. Jama, and R. Mao Molecular Diagnosis of Prader-Willi and Angelman Syndromes by Methylation-Specific Melting Analysis and Methylation-Specific Multiplex Ligation-Dependent Probe Amplification Clin. Chem., July 1, 2006; 52(7): 1276 - 1283. [Abstract] [Full Text] [PDF]
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T. Tabone, G. Sallmann, E. Webb, and R. G. H. Cotton Detection of 100% of mutations in 124 individuals using a standard UV/Vis microplate reader: a novel concept for mutation scanning Nucleic Acids Res., March 22, 2006; 34(6): e45 - e45. [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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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]
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 C. Parker, M. Omine, S. Richards, J.-i. Nishimura, M. Bessler, R. Ware, P. Hillmen, L. Luzzatto, N. Young, T. Kinoshita, et al. Diagnosis and management of paroxysmal nocturnal hemoglobinuria Blood, December 1, 2005; 106(12): 3699 - 3709. [Full Text] [PDF]
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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]
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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. Beutler and C. West Hematologic differences between African-Americans and whites: the roles of iron deficiency and {alpha}-thalassemia on hemoglobin levels and mean corpuscular volume Blood, July 15, 2005; 106(2): 740 - 745. [Abstract] [Full Text] [PDF]
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 E. Schutz, H. B. Urnovitz, L. Iakoubov, W. Schulz-Schaeffer, W. Wemheuer, and B. Brenig Bov-tA Short Interspersed Nucleotide Element Sequences in Circulating Nucleic Acids from Sera of Cattle with Bovine Spongiform Encephalopathy (BSE) and Sera of Cattle Exposed to BSE Clin. Vaccine Immunol., July 1, 2005; 12(7): 814 - 820. [Abstract] [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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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]
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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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C. P. Vaughn and K. S. J. Elenitoba-Johnson High-Resolution Melting Analysis for Detection of Internal Tandem Duplications J. Mol. Diagn., August 1, 2004; 6(3): 211 - 216. [Abstract] [Full Text]
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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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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]
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M. Liew, R. Pryor, R. Palais, C. Meadows, M. Erali, E. Lyon, and C. Wittwer Genotyping of Single-Nucleotide Polymorphisms by High-Resolution Melting of Small Amplicons Clin. Chem., July 1, 2004; 50(7): 1156 - 1164. [Abstract] [Full Text] [PDF]
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S. Giglio, P. T. Monis, and C. P. Saint Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR Nucleic Acids Res., November 15, 2003; 31(22): e136 - e136. [Abstract] [Full Text] [PDF]
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