Lan-Szu Chou, PhD, 1 Elaine Lyon, PhD, 1,2 and Carl T. Wittwer, MD, PhD 1,2* Abstract

Transcription

1 Basic Science / CFTR GENE SCANNING BY MELTING A Comparison of High-Resolution Melting Analysis With Denaturing High-Performance Liquid Chromatography for Mutation Scanning Cystic Fibrosis Transmembrane Conductance Regulator Gene as a Model Lan-Szu Chou, PhD, 1 Elaine Lyon, PhD, 1,2 and Carl T. Wittwer, MD, PhD 1,2* Key Words: CFTR; High-resolution melting analysis; Mutation scanning; Denaturing high-performance liquid chromatography; dhplc DOI: /BF3MLJN8J527MWQY Abstract High-resolution melting analysis (HRMA) was compared with denaturing high-performance liquid chromatography (dhplc) for mutation scanning of common mutations in the cystic fibrosis transmembrane conductance regulator gene. We amplified (polymerase chain reaction under conditions optimized for melting analysis or dhplc) 26 previously genotyped samples with mutations in exons 3, 4, 7, 9, 10, 11, 13, 17b, and 21, including 20 different genotypes. Heterozygous mutations were detected by a change in shape of the melting curve or dhplc tracing. All 20 samples with heterozygous mutations studied by both techniques were identified correctly by melting (100% sensitivity), and 19 were identified by dhplc (95% sensitivity). The specificity of both methods also was good, although the dhplc traces of exon 7 consistently revealed 2 peaks for wild-type samples, risking false-positive interpretation. Homozygous mutations could not be detected using curve shape by either method. However, when the absolute temperatures of HRMA were considered, G542X but not F508del homozygotes could be distinguished from wild type. HRMA easily detected heterozygotes in all single nucleotide polymorphism (SNP) classes (including A/T SNPs) and 1- or 2-basepair deletions. HRMA had better sensitivity and specificity than dhplc with the added advantage that some homozygous sequence alterations could be identified. HRMA has great potential for rapid, closedtube mutation scanning. Identifying genetic alterations throughout an entire gene is necessary in many complex inherited diseases. For example, the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) consists of 27 exons. 1 Each exon differs in size, and mutations are distributed throughout the entire gene. Some of the mutations, such as F508del in exon 10, are associated with severe phenotypes, and some are associated with mild symptoms. 2-5 More than 1,000 mutations have been identified in this gene. 4 To fully analyze such a broad mutation spectrum, a powerful mutation-screening tool is needed to scan the exons and intronic splice regions. Several mutation scanning methods are available, including denaturing gradient gel electrophoresis, 6 conformation sensitive gel electrophoresis, 7 temperature gradient capillary electrophoresis, 8-10 denaturing high performance liquid chromatography (dhplc), 11,12 and high-density oligonucleotide arrays. 12 These methods have varying sensitivities and require intensive labor or sophisticated instruments to perform analysis. In contrast, melting curve analysis is easy to perform and is a good candidate for mutation scanning. Conventional melting curve analysis uses fluorescently labeled probes or primers to detect sequence variants within a limited region Previous attempts to use DNA dyes for mutation scanning of entire amplicons have required processing steps after polymerase chain reaction (PCR), such as addition of urea 17 or product purification and addition of high dye concentrations. 18 The commonly used dye SYBR Green I limits melting resolution because of dye redistribution during melting, a property not observed with some dyes recently synthesized specifically for high-resolution melting (LCGreen I dyes, Idaho Technology, Salt Lake City, UT). LCGreen I has been used for 330 Am J Clin Pathol 2005;124: DOI: /BF3MLJN8J527MWQY

2 Basic Science / ORIGINAL ARTICLE rapid genotyping, 19,20 and a feasibility study of its use in scanning with a plasmid model has recently been published. 21 To evaluate the usefulness of high-resolution melting for gene scanning, 9 exons with 20 different genotypes in the CFTR gene were selected as targets. These exons covered some of the most common mutations of the CFTR gene. 22,23 Sequence alterations were detected by comparing the PCR product melting profiles or dhplc traces of mutation positive with wild-type samples. amplifying a 292-base-pair (bp) fragment and mixing (1:1) with wild-type amplicon. Informative amplicons for each of the 26 samples were amplified, analyzed, and compared with the appropriate wild-type control samples. All samples were run in duplicate. Wild-type control samples for each exon were sequenced and confirmed against published sequences ( Gene ID, ENSG ). Bidirectional sequencing was done using the BigDye Terminator chemistry (Applied Biosystems, Foster City, CA) on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Materials and Methods Sample Source and Study Design Eleven commercially genotyped samples were obtained from Coriell Cell Repositories, Coriell Institute for Medical Research, Camden, NJ (Y122X, R334W, R347P, A455E, I507del, F508del, F508C, G542X/G542X, R553X, R560T, and M1101K). In addition, 15 clinical samples of known genotype determined by the oligo ligation assay (OLA, Celera Diagnostics, Alameda, CA) were selected for analysis after deidentification and institutional review board approval. These 26 samples included 20 different genotypes with mutations associated with 9 different exons Table 1. Genomic DNA from clinical samples was extracted by the MagNA Pure LC (Roche Diagnostics, Indianapolis, IN). One homozygous Coriell sample (M1101K) was converted into a heterozygous template by Table 1 Mutations Analyzed in the Study Mutation Scanning by High-Resolution Melting Analysis Primers for exons 3, 4, 7, 9, 10, 11, 13, 17b, and 21 of the CFTR gene were designed as described previously 10 using Primer3 software. 24 An internal reverse primer was used to segment exon 13 (5'-GGGAGTCTTTTGCACAATGG-3'). PCR was performed in 25-µL reaction volumes using puretaq Ready-To-Go PCR beads (Amersham Biosciences, Piscataway, NJ) with 1 LCGreen I (Idaho Technology) and 100 to 150 ng of DNA per reaction. PCR was performed in the LightCycler (Roche Diagnostics) with an initial denaturation at 95 C for 5 minutes, followed by 40 cycles of 94 C for 2 seconds (transition rate, 20 C per second), 55 C for 10 seconds (transition rate, 15 C per second), and 72 C for 20 seconds (transition rate, 2 C per second). Following amplification, PCR products were denatured at 95 C for 5 minutes and rapidly cooled to 40 C (transition rate, 20 C per second) to form heteroduplexes. Position From 5' Exon (or Intron) Genotype * No. of Samples Nucleotide Change SNP Class End/Amplicon Size (bp) 3 394delTT 1 Del 132/234 4 R117H 1 G A 1 83/270 Y122X 1 T A 4 99/270 I148T 2 T C 1 176/270 Intron G T 2 233/270 7 R334W 1 C T 1 208/345 R347P 1 G C 3 248/345 9 A455E 2 C A 2 155/ I507del 1 Del 171/292 F508del 3 Del 174/292 F508del/F508del 1 Del 174/292 F508C 1 T G 2 175/ G542X 1 G T 2 90/175 G542X/G542X 1 G T 2 90/175 G551D 1 G A 1 118/175 R553X 2 C T 1 123/175 R560T 1 G C 3 145/ delA 1 Del 356/458 17b M1101K 1 T A 4 196/ N1303K 1 C G 3 175/250 bp, base pairs; SNP, single nucleotide polymorphism. * All genotypes were heterozygous except homozygous F508del and G542X. Classification based on Liew et al delTT, deletion of 2 bp in complementary DNA (cdna) from position 394; I507del, deletion of 3 bp between cdna positions 1648 and 1653; F508del, deletion of 3 bp between cdna positions 1652 and 1655; 2184delA, deletion of an A in cdna position 2184 ( Am J Clin Pathol 2005;124: DOI: /BF3MLJN8J527MWQY 331

3 Chou et al / CFTR GENE SCANNING BY MELTING PCR products were analyzed in the HR-1 (Idaho Technology), a single-capillary high-resolution melting instrument that collects approximately 50 data points per 1 C. 19 Data were acquired from 65 C to 95 C at a transition rate of 0.3 C per second and analyzed using version 1.1 software (Idaho Technology). In general, melting curves were normalized (between 0% and 100% fluorescent intensity), temperatureadjusted (superimposing the temperature axis of each curve over a certain fluorescence interval), and compared with wildtype control samples as previously described. 19 The normalization and temperature-adjustment steps allow visual comparison of melting curve shapes. Samples were called heterozygotes if the melting curve shape was different from that of the wildtype control samples. In some cases, temperature adjustment was not performed to see whether homozygous sequence changes could be identified. Predicted melting temperature (T m ) calculations were performed as previously described. 25 Stability maps were obtained using MELT94 software (available at Mutation Scanning by dhplc Twenty-two of the samples (all except 394delTT, Y122X, 2184delA, and M1101K) also were amplified under conditions optimized for dhplc with the same primers (Transgenomic, Omaha, NE). Each 50-µL PCR contained 2.5 U of Optimase polymerase (Transgenomic), 200 µmol/l of each deoxynucleoside triphosphate, 1 reaction buffer (Transgenomic), 0.4 µmol/l of each primer, 1.5 mmol/l of magnesium sulfate, and 50 to 100 ng of DNA. PCR was performed in a PTC-200 thermocycler (MJ Research, Reno, NV) by heating 5 minutes at 95 C, followed by 40 cycles of 30 seconds at 94 C, 60 seconds at 55 C, 60 seconds at 72 C, and a final extension at 72 C for 5 minutes. After amplification, heteroduplexes were formed by slow cooling (95 C for 5 minutes followed by cooling from 95 C to 25 C in 40 minutes) and analyzed directly by dhplc (Transgenomic WAVE system with a DNASep High Throughput cartridge). PCR products were eluted from the cartridge by a linear acetonitrile gradient with a constant flow rate at 1.5 ml/min. Wavemaker 4.1 software (Transgenomic) was used to determine the predicted optimal column temperature for each exon, and additional empirical optimization was performed if necessary. Detailed running parameters of the WAVE system are listed in Table 2. Results We scanned 16 known CFTR mutations in 6 exons of 22 samples by high-resolution melting and dhplc. For each method, all exons were amplified using 1 PCR program, and single PCR products were confirmed for each exon on a 2% agarose gel (data not shown). The melting curve and dhplc traces of the amplified samples were compared with wild-type samples to assess the sensitivity and specificity of each method. Of 20 heterozygous samples, all were detected by melting and 19 by dhplc for sensitivities of 100% and 95%, respectively. Homozygous mutations F508del and G542X could not be identified by melting analysis or dhplc when only curve shapes were compared. However, when melting curve position and shape were considered, G542X homozygotes could be identified by high-resolution melting. The exon 4 amplicon included 4 heterozygous single nucleotide substitutions within a 270-bp product (Table 1). The melting curves of duplicate samples were highly reproducible (data not shown). The normalized, temperature-shifted curves of each heterozygous mutation were different from the wild-type control sample. The melting curves and dhplc traces of 3 of these heterozygous single nucleotide polymorphisms (SNPs) are shown in Figure 1. Mutations altering a wild-type G::C bp (R117H and 621+1) showed greater differences than those that altered a wild-type T::A bp (I148T). The dhplc traces for exon 4 were best resolved at 57.3 C after empirical optimization (Figure 1D). The sequenced wildtype control sample showed a single peak, whereas the and I148T traces had multiple peaks. However, the R117H trace was similar to the control sample, resulting in the single false-negative result for dhplc in our study (Figure 1D). Exon 7 was amplified as a longer PCR amplicon (345 bp) and included 2 single base heterozygotes, R334W and R347P. Figure 2A shows that the amplicon melted in 2 domains as predicted by theory (inset), one between 75 C and 80 C and another between 82 C and 87 C. Each melting domain was Table 2 Optimized Parameters Used in the Denaturing High-Performance Liquid Chromatography (WAVE System) * Amplicon Melting Flow Rate Gradient Time Starting Buffer B Time Exon (base pairs) (ml/min) (min) (%) Shift * The application type was rapid DNA for all exons. For proprietary information, see the text. 332 Am J Clin Pathol 2005;124: DOI: /BF3MLJN8J527MWQY

4 Basic Science / ORIGINAL ARTICLE normalized individually and temperature-shifted; the results are shown in Figure 2B (lower domain) and Figure 2C (upper domain). The melting curves of the wild-type and heterozygous samples are identical in the lower melting domain (Figure 2B), indicating homologous sequence within this region. In contrast, different melting shapes were observed for R334W and R347P in the higher melting domain (Figure 2C), as expected from the location of the mutations. The exon 7 PCR products did not resolve well by dhplc using the T m s predicted by the commercial software (54.2 C, 55.2 C, and 56.2 C). Adequate resolution was A C R117H het C::A T::G G::C het C::T A::G G::C obtained with a higher T m (61.1 C), a different detection threshold (time shift, 0.5), and a longer elution time (4.5 minutes). Under these conditions, R334W and R347P had elution peaks that were different from that for the wild-type and were identified correctly as heterozygotes Figure 2D. However, the wild-type sample also showed 2 peaks, suggesting a heterozygous sequence. Sequencing confirmed that the sample was homozygous and identical to the published wild type. Additional samples then were sequenced as wild type, amplified, and analyzed by dhplc with the same results. Such double peaks usually are interpreted as B D Absorbance (mv) I148T het C::A T::G T::A het I148T het R117H het Time (min) Figure 1 High-resolution melting and denaturing high-performance liquid chromatography (dhplc) analysis of exon 4 of the cystic fibrosis transmembrane conductance regulator gene. Melting data were normalized and temperature shifted as previously described. 19 A, Heterozygous (het) R117H and the wild-type () control sample. B, Heterozygous I148T and the control sample. C, Intronic heterozygous G/T and the control sample. D, The dhplc profile of heterozygous mutations in exon 4. Am J Clin Pathol 2005;124: DOI: /BF3MLJN8J527MWQY 333

5 Chou et al / CFTR GENE SCANNING BY MELTING A B C Base Pairs R334W het C::A T::G G::C R347P het C::C G::G Absorbance (mv) R347P het R334W het Time (min) Figure 2 High-resolution melting and denaturing high-performance liquid chromatography (dhplc) analysis of exon 7 of the cystic fibrosis transmembrane conductance regulator gene. Melting data were normalized and temperature shifted as previously described. 19 A, Melting profile of the wild-type () control sample (345 base pairs). Inset, The predicted stability map of the amplicon. B, Low melting domain (75 C-80 C) comparison of heterozygous (het) R334W, R347P, and the control sample. C, High melting domain (82 C-87 C) comparison of heterozygous R334W, R347P, and the control sample. D, The dhplc profile of the homozygous, heterozygous R334W, and heterozygous R347P genotypes. D false-positives, decrease the specificity of mutation scanning, and result in unnecessary sequencing. Additional mutations in exons 9, 10, 11, and 21 included 7 heterozygous SNPs (A455E, F508C, G542X, G551D, R553X, R560T, and N1303K) and 2 heterozygous 3-base deletions (I507del and F508del). All were detected easily by high-resolution melting and dhplc scanning without falsenegative or false-positive concerns (data not shown). Examples of dhplc traces and melting profiles are shown in Figure 3 and Figure 4, respectively. Homozygous sequence alterations were difficult to distinguish from wild type by either method. The dhplc traces of homozygous G542X and F508del mutations were the same as wild-type control samples (Figure 3). In contrast, high-resolution melting analysis could distinguish some but not all homozygous sequence changes. G542X but not F508del homozygotes could be discerned from their wild types if curve position (absolute temperature) instead of curve shape was used for comparison. Nearest-neighbor calculations predict that the T m between wild type and G542X homozygotes is 334 Am J Clin Pathol 2005;124: DOI: /BF3MLJN8J527MWQY

6 Basic Science / ORIGINAL ARTICLE A B Absorbance (mv) G542X het G542X hom Time (min) 0.76 C. Analysis of 5 different wild types and the single available G542X homozygote revealed a shifted curve position (Figure 4), suggesting that absolute temperature differences may be used to identify homozygous sequence alterations. However, the predicted T m between wild type and F508del homozygotes is very small (0.01 C). When 5 different F508del homozygotes and 5 different wild-type samples were compared, a consistent difference in curve position between genotypes was not observed (data not shown). Four additional mutations were scanned only by high-resolution melting. Y122X and M1101K are both class 4 SNPs 20 (A/T heterozygotes), a class otherwise absent from our study. High-resolution melting analysis of these A/T heterozygotes showed clear separation from their respective control samples Figure 5A and Figure 5B. The common 3-bp deletion (F508del) was easy to identify when heterozygous (data not shown). Clear detection of even smaller heterozygous deletions is shown in Figure 5C (2184delA) and Figure 5D (394delTT). Absorbance (mv) F508del het F508del hom Time (min) Figure 3 Denaturing high-performance liquid chromatography analysis of mutations G542X (exon 11) and F508del (exon 10) of the cystic fibrosis transmembrane conductance regulator gene. A, Elution patterns of the wild-type (), heterozygous (het) G542X, and homozygous (hom) G542X samples. B, Elution patterns of the, heterozygous F508del, and homozygous F508del G542X het G542X hom Figure 4 High-resolution melting analysis of exon 11. Melting curves shown are 5 wild-type () samples, 1 G542X heterozygote (het), and 1 G542X homozygote (hom). Melting curves were not temperature shifted. Discussion Fluorescent melting analysis often is used in conjunction with real-time PCR to identify PCR products 26 or to genotype loci with hybridization probes. 14,27 Recently, high-resolution melting analysis with saturating DNA dyes has been proposed as a scanning tool to identify heteroduplex PCR products. 21 Apparent advantages include a closed-tube method with no need for processing or separation and speed of analysis (1-2 minutes). We compared this new technique with dhplc, one of the more commonly used platforms for mutation scanning. dhplc previously has been used for scanning the CFTR gene Analysis by dhplc of 6 exons of the CFTR gene was performed using predicted column temperatures with empiric optimization when necessary. In our hands, all heterozygous mutations were detected except R117H (exon 4), resulting in a heterozygote detection sensitivity of 95%. In comparison, Ravnik- Glavac et al 28 reported correct detection of heterozygous Am J Clin Pathol 2005;124: DOI: /BF3MLJN8J527MWQY 335

7 Chou et al / CFTR GENE SCANNING BY MELTING A B C Y122X het A::A T::T T::A delA het R117H with an overall heterozygote detection sensitivity of 90% when a single, predicted column temperature was used for each exon. In their study, sensitivity was increased to 100% by empirical optimization of the column temperature. In addition to occasional false-negatives, false-positives also were a concern with dhplc in our hands. Wild-type profiles of exon 7 showed 2 peaks. Sequencing of multiple wildtype samples confirmed that this pattern was not caused by heterozygous samples or contaminants. 31 Exon 4 (Figure 1) and exon 7 (Figure 2) showed multiple melting transitions, possibly explaining the sensitivity and specificity limitations of dhplc M1101K het A::A T::T T::A delTT het Figure 5 High-resolution melting detection of heterozygous A/T single nucleotide polymorphisms and small (1-2 base pair) deletions. Melting data were normalized and temperature shifted as previously described. 19 A, Heterozygous (het) Y122X (exon 4) and the wild-type () control sample. B, Heterozygous M1101K (exon 17b) and the control sample. C, Heterozygous 2184delA (exon 13) and the control sample. D, Heterozygous 394delTT (exon 3) and the control sample. D Multiple column temperatures might be needed to detect heterozygotes in different melting domains. 28 Furthermore, the widely separated domains of exon 7 might be the cause of the double peaks in the dhplc trace. As expected, both homozygous mutations tested were not detected by dhplc. High-resolution melting analysis is similar to dhplc in that both methods identify heteroduplexes by their lower thermal stability. However, unlike dhplc, melting analysis scans through a range of temperatures rather than depending on a specific temperature that requires optimization. To discriminate differences at single base resolution, saturating DNA 336 Am J Clin Pathol 2005;124: DOI: /BF3MLJN8J527MWQY

8 Basic Science / ORIGINAL ARTICLE dyes and high-resolution melting instruments are required, as opposed to conventional real-time instrumentation. 16,20 Melting analysis detected all heterozygous mutations tested (100% sensitivity). Heterozygous samples that were compared with an T::A wild type (Figure 1B) showed less deviation than samples compared with a G::C wild type (Figures 1A and 1C). This finding also is supported by a previous study using engineered plasmids, 21 in which the only false-negative scanning results were obtained with wild-type T::A pairs. We suggest the following explanation, considering SNPs that exchange an T::A pair with a G::C pair (type 1 and type 2 SNPs 20 ). When compared with a G::C wild type, amplification of heterozygotes results in less stable heteroduplexes and less stable T::A homoduplexes. In contrast, when compared with an T::A wild type, less stable heteroduplexes are partly offset by more stable C::G homoduplexes. All classes of SNPs, 20 including all possible heterozygous combinations of bases (A/C, A/G, A/T, C/G, C/T, and G/T), were detected by high-resolution melting. Furthermore, deletions of 1 to 3 bp were easy to identify. These findings are supported by previous work on plasmids 21 and genomic targets In the present study, different heterozygotes within the same exon could be distinguished easily from each other by different curve shapes (Figures 1 and 2, and data not shown). However, even though any heterozygote is easy to discriminate from wild type, it is unlikely that all mutations within an exon are distinguishable from each other. For example, in addition to the R334W mutation studied, 6 more mutations have been reported for this amino acid (R334C, E, H, K, L, Q). 35 It would be interesting to compare the melting curves of all 7 R334 mutations to see how many can be distinguished from each other. It generally is accepted that routine dhplc scanning does not detect homozygous sequence alterations. The situation for melting analysis is less clear. In 84% of human SNPs, the T m difference between wild type and homozygous mutant in small amplicons is greater than 0.5 C, 20 a difference easily detectable by high-resolution melting. Only 4% of human SNPs have nearest-neighbor symmetry with no predicted difference in T m between homozygotes. Homozygous SNP detection has been reported in PCR products up to 544 bp. 19 In the present study, dhplc missed the G542X homozygote, but melting analysis was able to identify the homozygous change using T m differences. Contrary to scanning for heterozygotes in which curve shape is paramount, identifying homozygous variants requires consideration of the absolute temperature or position along the temperature axis. The potential to detect most homozygous variants is an advantage of melting analysis over other heteroduplex-based methods. Both dhplc and melting analysis missed the F508del homozygous sample. The predicted T m is so small it is unlikely that any method based on thermal stability would detect the difference between F508del and wild-type homozygotes. One solution is to mix the samples before or after PCR to create artificial heterozygotes. 20 However, mixing before PCR requires accurate quantification, and mixing after PCR requires opening the system, negating a major advantage of closed-tube melting analysis. Alternatively, unlabeled oligonucleotide probes 25 can be added to the mixture before PCR. From 1 melting curve, analysis of the PCR product transition should allow scanning the entire PCR product, and the unlabeled probe transition should define the genotype. Saturating DNA dyes such as LCGreen I should be able to monitor both transitions from 1 melting curve. High-resolution melting analysis, similar to other scanning techniques, requires accurate PCR amplification. Any doublestranded molecules (such as primer dimers or nonspecific products) are capable of binding dye and will influence melting profiles. Sometimes PCR artifacts can be discriminated when the aberrant products melt at temperatures different from the desired amplicon. However, it is best to avoid potential interference by carefully optimizing PCR and/or using hot-start techniques. The sensitivity and specificity of high-resolution melting over all possible single base mismatches has been studied with engineered plasmids of 40%, 50%, and 60% GC content. 21 Sensitivity and specificity were 100% for amplicons less than 400 bp but dropped to 96% sensitivity and 99% specificity from 400 to 1,000 bp. To avoid errors, long exons may need to be segmented into multiple amplicons for scanning. High-resolution instruments are necessary because the resolution of current realtime instruments is limited. 16,20 Resolution also depends on careful control of sample evaporation, and heteroduplex formation is dependent on the Mg++ concentration. 16 High-resolution melting analysis is a rapid (1-2 minutes), closed-tube method that is at least as accurate as other scanning techniques that require heteroduplex separation. Different melting patterns correlate with different mutations, and many homozygous mutations can be identified. Melting analysis provides a convenient way to detect mutations in a large multiexon gene without performing full gene sequencing. However, before clinical implementation, more comprehensive studies are needed to define the limits of high-resolution melting for mutation screening. Furthermore, current instrumentation is limited to analysis of 1 sample at a time. Although the throughput is reasonable at 45 samples per hour, the development of microtiter platforms would greatly simplify handling and further increase throughput. From the 1 Institute for Clinical and Experimental Pathology, ARUP Laboratories; and 2 Department of Pathology, University of Utah School of Medicine, Salt Lake City. Supported by the Institute for Clinical and Experimental Pathology, ARUP Laboratories. Address reprint requests to Dr Chou: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT Acknowledgments: We thank Idaho Technology, Salt Lake City, UT, for placement of high-resolution melting instrument and Am J Clin Pathol 2005;124: DOI: /BF3MLJN8J527MWQY 337

9 Chou et al / CFTR GENE SCANNING BY MELTING reagents; Transgenomic, Omaha, NE, for use of the dhplc system; and Michael Liew, PhD, for useful discussions. Aspects of high-resolution melting analysis are licensed from the University of Utah to Idaho Technology. * Dr Wittwer holds equity interest in Idaho Technology. References 1. Riordan JR, Rommens JM, Kerem B, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989;245: Noone PG, Knowles MR. CFTR-opathies : disease phenotypes associated with cystic fibrosis transmembrane regular gene mutations. Respir Res. 2001;2: Rohlfs EM, Zhou Z, Sugarman EA, et al. The I148T CFTR allele occurs on multiple haplotypes: a complex allele is associated with cystic fibrosis. Genet Med. 2002;4: Ratjen F, Döring G. Cystic fibrosis. Lancet. 2003;361: Lee JH, Choi JH, Namkung W, et al. A haplotype-based molecular analysis of CFTR mutations associated with respiratory and pancreatic diseases. 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