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DNA Diagnostics by Surface-Bound Melt-Curve Reactions

2007; Elsevier BV; Volume: 9; Issue: 1 Linguagem: Inglês

10.2353/jmoldx.2007.060057

1943-7811

Linda Strömqvist Meuzelaar, Katie L. Hopkins, E. Liébana, Anthony J. Brookes,

Molecular Biology Techniques and Applications

Melting-curve procedures track DNA denaturation in real time and so provide an effective way of assessing sequence variants. Dynamic allele-specific hybridization (DASH) is one such method, based on fluorescence, which uses heat to denature a single allele-specific probe away from one strand of a polymerase chain reaction product attached to a solid support. DASH is a proven system for research genotyping, but here we evaluate it for DNA diagnostics under two scenarios. First, for mutation scanning (resequencing), a human genomic sequence of 97 bp was interrogated with 15 probes tiled with 12-base overlaps, providing up to fourfold redundancy per base. This test sequence spanned three high-frequency single nucleotide polymorphisms, all of which were correctly revealed in 16 individuals. Second, to score multiple different mutations in parallel, 18 alterations in the gyrA gene of Salmonella were assessed in 62 strains by using wild-type- and mutation-specific probes. Both experiments were performed in a blinded manner, and all results were confirmed by sequencing. All DNA variants were unambiguously resolved in every sample, with no false-positive or false-negative signals across all of the investigations. In conclusion, DASH performs accurately and robustly when applied to DNA diagnostic challenges, including mutation scoring and mutation scanning. Melting-curve procedures track DNA denaturation in real time and so provide an effective way of assessing sequence variants. Dynamic allele-specific hybridization (DASH) is one such method, based on fluorescence, which uses heat to denature a single allele-specific probe away from one strand of a polymerase chain reaction product attached to a solid support. DASH is a proven system for research genotyping, but here we evaluate it for DNA diagnostics under two scenarios. First, for mutation scanning (resequencing), a human genomic sequence of 97 bp was interrogated with 15 probes tiled with 12-base overlaps, providing up to fourfold redundancy per base. This test sequence spanned three high-frequency single nucleotide polymorphisms, all of which were correctly revealed in 16 individuals. Second, to score multiple different mutations in parallel, 18 alterations in the gyrA gene of Salmonella were assessed in 62 strains by using wild-type- and mutation-specific probes. Both experiments were performed in a blinded manner, and all results were confirmed by sequencing. All DNA variants were unambiguously resolved in every sample, with no false-positive or false-negative signals across all of the investigations. In conclusion, DASH performs accurately and robustly when applied to DNA diagnostic challenges, including mutation scoring and mutation scanning. In general terms, DNA diagnostic challenges involve testing the DNA of an individual for sequence variations that could be relevant to the genetic etiology of a particular phenotype in that individual. Applications could range from scoring single base alterations (eg, known pathogenic mutations) in one or a few individuals, through to population-level screening for known or novel changes that might cause disease. At this latter extreme, the challenge overlaps with research into the genetic basis of disease. In practice, most real-world DNA diagnostic activities involve testing particularly likely disease genes in small numbers of individuals to answer one or two clinically relevant questions, are any of a limited set of known pathogenic mutations present in that gene (mutation scoring), and/or are any suspicious changes present anywhere in that gene (mutation scanning)? In addition, chromosome level and structural variation analyses may be conducted, but such investigations are beyond the focus of this current report. The technologies used for mutation scoring and mutation scanning are mostly distinct, but it would be preferable if diagnostics procedures could be applied equally well to both challenges, using standardized and convenient reaction formats. Such a truly generic DNA diagnostics system is probably still some way off, but it is nevertheless desirable that current method development efforts emphasize solutions that are as flexible as possible. Toward that goal, many factors might be considered, such as: 1. The assay target—Is the objective to score one critical bp, to assess all suspect sites in one or several genes, to resequence several genes/genome regions, or to test extremely large numbers of bases to report on a complete-genome (at least to some degree of depth)? 2. The result precision—Will it be sufficient to determine merely whether a sample DNA is the same as a reference sequence, whereas in other scenarios, the precise location and nature of extant changes relative to the reference must be elucidated? 3. The reaction chemistry—How important are factors such as the need for standard run conditions versus assay-specific optimization, the cost per result, and the speed of data generation? 4. The reaction format—How desirable is it that the procedure is homogenous (sealed tube) rather than nonhomogenous or single-step rather than multistep in its design? 5. The required equipment—Will the method be run on generic or method-specific devices, how expensive and easy to use will those instruments be, and what throughput potential will they have? Given so many diverse method features to consider, it is hard to imagine that one technology will ever fully satisfy the most extreme requirements imposed by all these criteria. It is also likely that complex and consequentially temperamental solutions will probably not be the way forward because such technologies are more sensibly reserved for highly specialized applications. Instead, to progress toward a generic solution it will be most effective to use robust and elegant underlying reaction principles. In this respect it is, therefore, not surprising that straightforward DNA hybridization—the simplest and most direct way to assess a DNA sequence—is now being increasingly exploited in the field of DNA diagnostics. The power of hybridization for DNA diagnostics stems from the fact that subtle sequence changes impose substantial changes in duplex stability such that, when assays are suitably formatted, each and every DNA sequence change can be reliably detected by direct or indirect measurement of that duplex stability. No additional enzymes or processing steps are required, and assays can be kept simple, cheap, and convenient. Hybridization methods may be designed to detect (but not specify) any difference relative to a reference sequence—as in denaturing high-performance liquid chromatography,1Underhill PA Jin L Lin AA Mehdi SQ Jenkins T Vollrath D Davis RW Cavalli-Sforza LL Oefner PJ Detection of numerous Y chromosome biallelic polymorphisms by denaturing high performance liquid chromatography.Genome Res. 1997; 7: 996-1005Crossref PubMed Scopus (578) Google Scholar,2O'Donovan MC Oefner PJ Roberts SC Austin J Hoogendoorn B Guy C Speight G Upadhyaya M Sommer SS McGuffin P Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection.Genomics. 1998; 52: 44-49Crossref PubMed Scopus (289) Google Scholar denaturing and temperature gradient gel electrophoresis,3Myers RM Lumelsky N Lerman LS Maniatis T Detection of single base substitutions in total genomic DNA.Nature. 1985; 313: 495-498Crossref PubMed Scopus (302) Google Scholar4Noll WW Collins M Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique.Proc Natl Acad Sci USA. 1987; 84: 3339-3343Crossref PubMed Scopus (40) Google Scholar5Riesner D Steger G Zimmat R Owens RA Wagenhofer M Hillen W Vollbach S Henco K Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions.Electrophoresis. 1989; 10: 377-389Crossref PubMed Scopus (208) Google Scholar single-strand conformational polymorphism analysis,6Orita M Iwahana H Kanazawa H Hayashi K Sekiya T Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms.Proc Natl Acad Sci USA. 1989; 86: 2766-2770Crossref PubMed Scopus (3380) Google Scholar,7Makino R Yazyu H Kishimoto Y Sekiya T Hayashi K F-SSCP: fluorescence-based polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis.PCR Methods Appl. 1992; 2: 10-13Crossref PubMed Scopus (91) Google Scholar and high-resolution amplicon melting analysis.8Wittwer CT Reed GH Gundry CN Vandersteen JG Pryor RJ High-resolution genotyping by amplicon melting analysis using LCGreen.Clin Chem. 2003; 49: 853-860Crossref PubMed Scopus (1010) Google Scholar,9Gundry CN Vandersteen JG Reed GH Pryor RJ Chen J Wittwer CT Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes.Clin Chem. 2003; 49: 396-406Crossref PubMed Scopus (294) Google Scholar These procedures subject test samples to dynamically changing environments that transition from low to high stringency and thereby exploit duplex stability differences and enable underlying mutations to be revealed. This same dynamic melting concept has also been used to locate and identify individual base changes by tracking the melting behavior of short normal or mutant oligonucleotides (probes) hybridized in solution to one strand of a target fragment produced by polymerase chain reaction (PCR) amplification. Melting profiles from single-labeled probes10Lay MJ Wittwer CT Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR.Clin Chem. 1997; 43: 2262-2267PubMed Google Scholar11Vrettou C Traeger-Synodinos J Tzetis M Malamis G Kanavakis E Rapid screening of multiple β-globin gene mutations by real-time PCR on the LightCycler: application to carrier screening and prenatal diagnosis of thalassemia syndromes.Clin Chem. 2003; 49: 769-776Crossref PubMed Scopus (73) Google Scholar12Millward H Samowitz W Wittwer CT Bernard PS Homogeneous amplification and mutation scanning of the p53 gene using fluorescent melting curves.Clin Chem. 2002; 48: 1321-1328PubMed Google Scholar or dual-labeled probes13Afonina IA Reed MW Lusby E Shishkina IG Belousov YS Minor groove binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence.Biotechniques. 2002; 32: 940-949PubMed Google Scholar,14Belousov Y Welch RA Sanders S Mills A Kulchenko A Dempcy R Afonina IA Walburger DA Glaser CL Yadavalli S Vermeulen NMJ Mahoney W Single nucleotide polymorphism genotyping by two colour melting curve analysis using the MGB Eclipse probe system in challenging sequence environment.Hum Genomics. 2004; 1: 209-217PubMed Google Scholar can be recorded by their change in fluorescence during denaturation. Far higher throughput hybridization analyses are made possible by using array formats on solid surfaces—such as dot blot and related methods15Kafatos FC Jones CW Efstratiadis A Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure.Nucleic Acids Res. 1979; 7: 1541-1552Crossref PubMed Scopus (907) Google Scholar,16Saiki RK Walsh PS Levenson CH Erlich HA Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes.Proc Natl Acad Sci USA. 1989; 86: 6230-6234Crossref PubMed Scopus (802) Google Scholar—wherein static, high-stringency reactions are used to quantitatively assess probe-target binding. Such arrays have since been developed into microarray formats17Kennedy GC Matsuzaki H Dong S Liu WM Huang J Liu G Su X Cao M Chen W Zhang J Liu W Yang G Di X Ryder T He Z Surti U Phillips MS Boyce-Jacino MT Fodor SP Jones KW Large-scale genotyping of complex DNA.Nat Biotechnol. 2003; 21: 1233-1237Crossref PubMed Scopus (455) Google Scholar,18Matsuzaki H Dong S Loi H Di X Liu G Hubbell E Law J Berntsen T Chadha M Hui H Yang G Kennedy GC Webster TA Cawley S Walsh PS Jones KW Fodor SP Mei R Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays.Nat Methods. 2004; 1: 109-111Crossref PubMed Scopus (352) Google Scholar so that many thousand different dispersed target sites can be tested in parallel. The same technology has also enabled long contiguous runs of bases to be examined on so-called resequencing chips.19Hacia JG Resequencing and mutational analysis using oligonucleotide microarrays.Nat Genet. 1999; 21: 42-47Crossref PubMed Scopus (433) Google Scholar,20Mockler TC Ecker JR Applications of DNA tiling arrays for whole-genome analysis.Genomics. 2005; 85: 1-15Crossref PubMed Scopus (299) Google Scholar Unfortunately, the static conditions used in high-throughput array hybridization systems fail to distinguish all sequence changes in all contexts. Logically then, it would make sense to couple the generic utility and power of dynamic hybridization to the throughput capabilities provided by array-formatted assays. This has previously been explored in the dynamic allele-specific hybridization (DASH) research genotyping method, which uses heat to control probe-target denaturation21Howell WM Jobs M Gyllensten U Brookes AJ Dynamic allele-specific hybridization. A new method for scoring single nucleotide polymorphisms.Nat Biotech. 1999; 17: 87-88Crossref PubMed Scopus (235) Google Scholar22Prince JA Feuk L Howell WM Jobs M Emahazion T Blennow K Brookes AJ Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH): design criteria and assay validation.Genome Res. 2001; 11: 152-162Crossref PubMed Scopus (161) Google Scholar23Jobs M Howell WM Strömqvist L Mayr T Brookes AJ DASH-2: flexible, low-cost, and high-throughput SNP genotyping by dynamic allele-specific hybridization on membrane arrays.Genome Res. 2003; 13: 916-924Crossref PubMed Scopus (63) Google Scholar and electronic microchips (eg, Nanogen, San Diego, CA), which uses an electrical field to control probe-target denaturation.24Sosnowski RG Tu E Butler WF O'Connell JP Heller MJ Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control.Proc Natl Acad Sci USA. 1997; 94: 1119-1123Crossref PubMed Scopus (422) Google Scholar,25Gilles PN Wu DJ Foster CB Dillon PJ Chanock SJ Single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor microchips.Nat Biotechnol. 1999; 17: 365-370Crossref PubMed Scopus (200) Google Scholar The DASH system is further detailed in Figure 1, and our extensive experience with this method for genotyping single nucleotide polymorphisms (SNPs) (more than 4000 target sites examined, producing ∼2 million genotypes) has shown that single probes applied by this method reliably detect >95% of all sequence variants under standard run conditions, with a routine accuracy of ∼99.9%. DASH has been implemented as a microtiter plate-based version (DASH-121Howell WM Jobs M Gyllensten U Brookes AJ Dynamic allele-specific hybridization. A new method for scoring single nucleotide polymorphisms.Nat Biotech. 1999; 17: 87-88Crossref PubMed Scopus (235) Google Scholar) and as a membrane-based macroarray format that interrogates up to 10,000 samples per array (DASH-223Jobs M Howell WM Strömqvist L Mayr T Brookes AJ DASH-2: flexible, low-cost, and high-throughput SNP genotyping by dynamic allele-specific hybridization on membrane arrays.Genome Res. 2003; 13: 916-924Crossref PubMed Scopus (63) Google Scholar). DASH has also shown itself highly effective as a means to score insertion/deletion variants26Sawyer SL Howell WM Brookes AJ Scoring insertion-deletion polymorphisms by dynamic allele-specific hybridization.Biotechniques. 2003; 35: 292-298PubMed Google Scholar and, via its quantitative capabilities, has recently enabled us to reveal the existence of extensive copy number variation in the human genome.27Fredman D White SJ Potter S Eichler EE Den Dunnen JT Brookes AJ Complex SNP-related sequence variation in segmental genome duplications.Nat Genet. 2004; 36: 861-866Crossref PubMed Scopus (193) Google Scholar Given the proven capabilities of DASH in the SNP genotyping arena, and the considerable needs of DNA diagnostics, we reasoned that DASH might be effective as an advanced hybridization platform for DNA diagnostics. We therefore explored this possibility, both in the context of scanning for mutations in a human genomic sequence and for the simultaneous scoring of many commonly mutated sites in quinolone-resistant Salmonella strains. This latter application represents a significant real-world example of troublesome bacterial antibiotic resistance. Quinolone resistance often arises because of spontaneous point mutations that cause amino acid substitutions within the topoisomerase subunits, often in combination with decreased expression of outer membrane porins or overexpression of multidrug efflux pumps.28Hopkins KL Davies RH Threlfall EJ Mechanisms of quinolone resistance in Escherichia coli and Salmonella: recent developments.Int J Antimicrob Agents. 2005; 25: 358-373Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar29Randall LP Woodward MJ Multiple antibiotic resistance (mar) locus in Salmonella enterica serovar typhimurium DT104.Appl Environ Microbiol. 2001; 67: 1190-1197Crossref PubMed Scopus (51) Google Scholar30Giraud E Cloeckaert A Kerboeuf D Chaslus-Dancla E Evidence for active efflux as the primary mechanism of resistance to ciprofloxacin in Salmonella enterica serovar typhimurium.Antimicrob Agents Chemother. 2000; 44: 1223-1228Crossref PubMed Scopus (206) Google Scholar We report the findings here, leading to the conclusion that both DASH-1 and a newly adapted version of DASH-2 are highly accurate and useful for DNA diagnostic applications. Human DNA samples from 16 unrelated Swedish females were prepared by standard phenol-chloroform extraction procedures. Salmonella DNA was prepared from 62 Salmonella enterica isolates received at the Health Protection Agency Centre for Infections Salmonella Reference Unit between 1991 and 2002. DNA from these strains was prepared from 24-hour cultures using a Qiagen DNeasy tissue kit (Qiagen, West Sussex, UK) according to the manufacturer's instructions. In addition, DNA from seven of the strains were also prepared using a simpler method: a single colony was resuspended in 100 μl of distilled water with 15% (w/v) Chelex 100 molecular biology grade resin (Bio-Rad, Hertfordshire, UK) and boiled for 10 minutes. The cell suspension was then centrifuged for 5 minutes at 13,000 rpm, and the supernatant removed and stored at −20°C until required. Oligonucleotides (PCR primers and probes) were obtained from Thermo Electron GmbH (Ulm, Germany) and Biomers.net GmbH (Ulm, Germany). One of the primers in a primer pair contained a 5′-biotin group, and all probes were labeled with a 3′-ROX moiety. Primers were designed using the OLIGO software (Molecular Biology Insights, Inc., Cascade, CO). Basic rules for primer design were primer length restricted to 20 to 24 bp, primer Tm difference 200 bp), thermal cycling consisted of an initial activation step of 94°C for 10 minutes, followed by 40 cycles of 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 1 minute. To sequence the human genomic DNA fragment, a 300-bp fragment encompassing the 97-bp region of interest using the primers LSCAN-23F and LSCANb22R (Table 1). Fifty-μl PCR products were purified using a MinElute PCR purification kit (Qiagen) following the manufacturer's protocol and eluting the PCR products in water. One μl of each eluted product (∼25 ng) was used as template for cycle sequencing that used a BigDye 3.1 terminator kit (Applied Biosystems), using 0.16 μmol/L of either of the initial amplification primers in 20-μl reactions. Cycling conditions consisted of 96°C for 1 minute followed by 25 cycles of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 minutes using a 96-well MultiBlock system (Thermo Electron Corporation). Sequencing products were purified using DyeEx 2.0 spin columns (Qiagen) following the manufacturer's protocol, and the samples were finally sequenced on a 48-capillary 3730 DNA analyzer (Applied Biosystems). Sequence data for the Salmonella gyrA gene was generated from a 342-bp fragment amplified using primers P1 (5′-TGTCCGAGATGGCCTGAAGC-3′) and P2 (5′-TACCGTCATAGTTATCCACG-3′).31Griggs DJ Gensberg K Piddock LJ Mutations in gyrA gene of quinolone-resistant Salmonella serotypes isolated from humans and animals.Antimicrob Agents Chemother. 1996; 40: 1009-1013PubMed Google Scholar Fifty-μl PCR products were purified using a Unifilter 96-well microplate (Whatman, Middlesex, UK) following the manufacturer's protocol. Two μl of each eluted product was used as template for cycle sequencing using 10 pmol of primer P1 and a CEQ Dye Terminator cycle sequencing quick start kit (Beckman Coulter, Buckinghamshire, UK) in 20-μl reactions. Cycling conditions consisted of 30 cycles of 96°C for 20 seconds, 50°C for 20 seconds, and 60°C for 4 minutes using a 96-well MultiBlock system (Thermo Electron Corporation). Sequencing products were purified by following the Dye Terminator cycle sequencing quick start kit protocol and the samples analyzed using a CEQ 8000 DNA analysis system (Beckman Coulter) using the LFR-1 run conditions. Resulting sequencing data are available as supplemental material at . DASH-1 was conducted as described previously.21Howell WM Jobs M Gyllensten U Brookes AJ Dynamic allele-specific hybridization. A new method for scoring single nucleotide polymorphisms.Nat Biotech. 1999; 17: 87-88Crossref PubMed Scopus (235) Google Scholar In brief, PCR products were diluted 1:1 with HEN buffer (0.1 mol/L Hepes, 10 mmol/L ethylenediaminetetraacetic acid, and 50 mmol/L NaCl, pH 7.5), and 20 μl per well was bound to a 96-well streptavidin-coated microtiter plate. The solution was then removed, and the wells were rinsed once with 25 μl of 0.1 mol/L NaOH to elute the unbound (nonbiotinylated) strand of the PCR product. A 25-μl solution containing HEN plus 15 pmol allele-specific probes was added. The microtiter plate was sealed, heated to 85°C, and air-cooled to 25°C for ∼5 minutes, enabling the probe to hybridize to the bound PCR product (regardless of which alleles were present). The solution was replaced with HEN containing SYBR Green I dye (Molecular Probes/Invitrogen, Paisley, UK) at a 1:10,000 dilution. The plates were analyzed in a DASH instrument (Thermo Hybaid, although any Q-PCR machine would suffice), and fluorescence was recorded while heating from 35 to 85°C at a rate of 0.3°C/second. DASH-2 was conducted in a manner similar to that previously described,23Jobs M Howell WM Strömqvist L Mayr T Brookes AJ DASH-2: flexible, low-cost, and high-throughput SNP genotyping by dynamic allele-specific hybridization on membrane arrays.Genome Res. 2003; 13: 916-924Crossref PubMed Scopus (63) Google Scholar with a few changes that enabled the diagnostics application. PCR products were first transferred from a 384-well microtiter plate to a streptavidin-coated polypropylene membrane via centrifugation as previously described.32Jobs M Howell WM Brookes AJ Creating arrays by centrifugation.Biotechniques. 2002; 32: 1322-1329PubMed Google Scholar To achieve this, the membrane (DynaMetrix Ltd., Hertfordshire, UK, ) was premoistened in HE buffer (0.05 mol/L Hepes and 5 mmol/L ethylenediaminetetraacetic acid, pH 7.5) and placed over the open wells of the microtiter plate. The arrangement was compressed in a clamping device and centrifuged at 1500 rpm for 30 seconds in a suitable device (S20 rotor; B4i Jouan; Thermo Scientific). After binding at room temperature for 30 minutes, the clamped structure was inverted and briefly centrifuged to return the bulk fluid into the microtiter plate wells. The membranes were then rinsed once in a 0.1 mol/L NaOH bath for 2 minutes to remove nonbiotinylated PCR product strands and once in HE for neutralization. To apply different probes to distinct locations on the same membrane, 10 pmol/μl of appropriate probe solution in HE buffer was placed in the matching well of a 384-well plate, and this was transferred to cover the membrane area where PCR product had been bound using the same clamping device and centrifuge as described above. Excess probe solution was immediately transferred back to the wells of the plate. The membrane was then recovered from the clamp and placed in a sandwich of two 8 × 12-cm glass plates (slightly larger than the membrane) to form a hybridization chamber. This was heated to 85°C on a flat PCR block (PCR Express; Thermo Electron Corporation) and air-cooled to room temperature to assist probe annealing. A final rinse was performed in HE to remove excess probe. To execute the dynamic melt procedure, membranes were soaked for 1 hour in HE buffer containing a 1:20,000 dilution of supplied stock SYBR Green I dye (Molecular Probes), and they were then individually sandwiched between two glass plates and placed into a DASH-2 genotyping device (DynaMetrix Ltd.). Fluorescence images and feature intensity values were collected while heating the membrane assembly from 35 to 85°C (with a heating rate of 3°C/minute) by imaging every 0.5°C. Output fluorescence data files were imported into purpose-built software (DynaScore; DynaMetrix Ltd.). Using this tool, melt-curves were examined for each microtiter plate well (DASH-1) or array feature (DASH-2), and probe-target denaturation events were visualized by plotting negative derivatives curves of the fluorescence signal versus temperature. A single high-temperature peak indicated the sample was completely matched to the probe sequence. A single low temperature peak indicated a 1- or 2-bp mismatch compared with the probe sequence (two-base mismatches cause much larger decreases in melting peak temperature, and more than two-base mismatches fall below the temperature window examined). If peaks were seen at both temperatures, this indicated that two alleles were present in the PCR product, such as would occur with a human heterozygous sample. To reliably scan all bases in a gene for mutations, a method must be able to detect essentially all possible base changes in any sequence context, preferably under standard run conditions. To explore the ability of DASH to achieve this, we conducted a blinded experiment in