High-resolution Amplicon Melting Research Articles

Bacillus anthracis strain differentiation based on SNP and VNTR loci.

Bacillus anthracis is the anthrax causative agent. For its epidemiology, it is important not only to identify the etiological agent but also to determine the patterns of its evolution and spread. Modern methods of molecular biology make it possible to detect a number of genetic markers suitable for indicating and differentiating the strains of B. anthracis, including the loci arranged as variable number tandem repeats (VNTRs) and SNPs, one nucleotide-sized differences in the DNA sequence of the loci being compared. The objective of the present study was to examine the effectiveness of SNP analysis and PCR amplif ication of VNTR loci combined with the high-resolution amplicon melting analysis for identif ication and differentiation of the anthrax agent strains. In the study, seven strains of B. anthracis obtained from soil samples and animal carcasses were investigated using vaccine strain STI-1 as a reference. For molecular genetic characterization of these bacteria, analysis of 12 SNPs and variability analysis of eight VNTR loci were carried out. To detect the differences between the strains, their PCR product melting points were measured in the presence of the EvaGreen (Sintol, Russia) intercalating dye. For SNP detection, a PCR assay with double TaqMan probes was applied. It was found that the studied virulent strains, except for B. anthracis No. 1 and 3, could not be attributed to any phylogenetic subgroup of the anthrax agents. The proposed method made it possible to differentiate four out of the seven investigated strains. Strains No. 5–7 had identical SNP and HRM prof iles and, as a result, formed a single cluster. Our investigation has conf irmed that the proposed method can be successfully used for preliminary analysis of an epizootic situation in the case of anthrax.

Quadruplex Genotyping of F5, F2, and MTHFR Variants in a Single Closed Tube by High-Resolution Amplicon Melting

Multiplexed amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, asymmetric PCR, or allele-specific PCR; however, correct differentiation of homozygous mutant and wild-type samples by melting temperature (T(m)) analysis requires high-resolution melting analysis and controlled reaction conditions. We designed 4 amplicons bracketing the F5 [coagulation factor V (proaccelerin, labile factor)] 1691G>A, MTHFR (NADPH) 1298A>C, MTHFR 677C>T, and F2 [coagulation factor II (thrombin)] 20210G>A gene variants to melt at different temperatures by varying amplicon length and adding GC- or AT-rich 5' tails to selected primers. We used rapid-cycle PCRs with cycles of 19-23 s in the presence of a saturating DNA dye and temperature-correction controls and then conducted a high-resolution melting analysis. Heterozygotes were identified at each locus by curve shape, and homozygous genotypes were assigned by T(m). We blinded samples previously genotyped by other methods before analysis with the multiplex melting assay (n = 110). All samples were correctly genotyped with the exception of 7 MTHFR 1298 samples with atypical melting profiles that could not be assigned. Sequencing revealed that these 5 heterozygotes and 2 homozygotes contained the unexpected sequence variant MTHFR 1317T>C. The use of temperature-correction controls decreased the T(m) SD within homozygotes by a mean of 38%. Rapid-cycle PCR with high-resolution melting analysis allows simple and accurate multiplex genotyping to at least a factor of 4.

Identification of ryanodine receptor 1 single-nucleotide polymorphisms by high-resolution melting using the LightCycler 480 System

High-resolution melting (HRM) allows single-nucleotide polymorphism (SNP) detection/typing using inexpensive generic heteroduplex-detecting double-stranded DNA (dsDNA) binding dyes. Until recently HRM has been a post-PCR process. With the LightCycler 480 System, however, the entire mutation screening process, including post-PCR analysis, can be performed using a single instrument. HRM assays were developed to allow screening of the ryanodine receptor gene ( RYR1) for potential mutations causing malignant hyperthermia (MH) and/or central core disease (CCD) using the LightCycler 480 System. The assays were validated using engineered plasmids and/or genomic DNA samples that are either homozygous wild type or heterozygous for one of three SNPs that lead to the RyR1 amino acid substitutions T4826I, H4833Y, and/or R4861H. The HRM analyses were conducted using two different heteroduplex-detecting dsDNA binding dyes: LightCycler 480 HRM dye and LCGreen Plus. Heterozygous samples for each of the HRM assays were readily distinguished from homozygous samples with both dyes. By using engineered plasmids, it was shown that even homozygous sequence variations can be identified by using either small amplicons or the addition of exogenous DNA after PCR. Thus, the LightCycler 480 System provides a novel, integrated, real-time PCR/HRM platform that allows high throughput, inexpensive SNP detection, and genotyping based on high-resolution amplicon melting.

High-Resolution Melting for Accurate Assessment of DNA Methylation

Methylation of cytosine in DNA is an epigenetic mark that is important for genome stability, transcriptional regulation of endogenous genes, and permanent silencing of transposable elements and viral sequences. Methylcytosine, sometimes referred to as the 5th base of DNA, constitutes 3%–6% of all cytosines in the human genome and occurs almost exclusively within the context of CpG dinucleotides (1). DNA methylation patterns have been shown to correlate with various disease states, including inherited disorders and cancer, and the identification of aberrantly methylated genes is a promising strategy in research, diagnostics, and therapeutics (2). In the current issue of Clinical Chemistry , White et al. (3) report a simple closed-tube PCR assay that combines amplification of bisulfite-treated DNA with high-resolution amplicon melting for sensitive and high-throughput assessment of DNA methylation. One major problem inherent in gene-specific DNA methylation analysis is that methylation marks are erased during conventional PCR and cloning procedures because methylcytosine is replaced with cytosine. To circumvent this problem, DNA can be treated with sodium bisulfite, which converts unmethylated cytosines to uracil, whereas methylcytosine is protected against this modification (4). The bisulfite-modified DNA can be used as template in a standard PCR to amplify specific DNA sequences and examine their methylation content. The most accurate methylation profiling can be achieved by sequence analysis of the PCR products, which display methylcytosine as cytosine and unmethylated …

Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting

Amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, or allele-specific polymerase chain reaction. However, correct differentiation of homozygous mutant and wild-type samples by melting temperature (Tm) requires high-resolution melting and closely controlled reaction conditions. When three different DNA extraction methods were used to isolate DNA from whole blood, amplicon Tm differences of 0.03 to 0.39 degrees C attributable to the extractions were observed. To correct for solution chemistry differences between samples, complementary unlabeled oligonucleotides were included as internal temperature controls to shift and scale the temperature axis of derivative melting plots. This adjustment was applied to a duplex amplicon melting assay for the methylenetetrahydrofolate reductase variants 1298A>C and 677C>T. High- and low-temperature controls bracketing the amplicon melting region decreased the Tm SD within homozygous genotypes by 47 to 82%. The amplicon melting assay was 100% concordant to an adjacent hybridization probe (HybProbe) melting assay when temperature controls were included, whereas a 3% error rate was observed without temperature correction. In conclusion, internal temperature controls increase the accuracy of genotyping by high-resolution amplicon melting and should also improve results on lower resolution instruments.

Characterization of Aberrant Melting Peaks in Unlabeled Probe Assays

An unlabeled probe assay relies on a double-stranded DNA-binding dye to detect and verify target based on amplicon and probe melting. During the development and application of unlabeled probe assays, aberrant melting peaks are sometimes observed that may interfere with assay interpretation. In this report, we investigated the origin of aberrant melting profiles observed in an unlabeled probe assay for exon 10 of the RET gene. It was determined that incomplete 3' blocking of the unlabeled probe allowed polymerase-mediated probe extension resulting in extension products that generated the aberrant melting profiles. This report further examined the blocking ability of the 3' modifications C3 spacer, amino-modified C6, phosphate, inverted dT, and single 3' nucleotide mismatches in unlabeled probe experiments. Although no 3' blocking modifications in these experiments were 100% effective, the amino-modified C6, inverted dT, and C3 spacer provided the best blocking efficiencies (1% or less unblocked), phosphate was not as effective of a block (up to 2% unblocked), and single nucleotide mismatches should be avoided as a 3' blocking modification.

DNA Diagnostics by Surface-Bound Melt-Curve Reactions

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.

Instrument Comparison for Dna Genotyping by Amplicon Melting

DNA melting analysis for the identification of sequence is increasingly used in molecular diagnostics. Recent advances in DNA melting analysis, including high-resolution instrumentation and specialized fluorescent DNA-binding dyes, allow genotyping by whole amplicon melting without probes. With the popularity of melting analysis as a diagnostic tool, there is a need to characterize the ability of commercially available real-time PCR instruments to perform high-resolution amplicon melting analyses. Four real-time instruments varying in sample format, throughput, and heat transfer (Cepheid's SmartCycler, Idaho Technology's HR-1, and Roche's LightCycler 1.2 and LightCycler 2.0) were evaluated for their ability to differentiate homozygous genotypes at the human β-globin sickle cell locus. The melting transition was monitored by including the dye LCGreen Plus in the PCR, and the data were uniformly analyzed with custom in-house software. The wild-type and mutant homozygous genotypes differed by a theoretical Tm of 0.09°C and were best discriminated by the high-resolution HR-1 instrument. All instruments could identify a double single nucleotide polymorphism heterozygote by the heteroduplexes formed. However, signal-to-noise ratios varied from 260 to 3500, suggesting that melting instrument design (data acquisition, data density, thermal control) determines the accuracy of genotyping by amplicon melting. (JALA 2006;11:273-7)

Genotyping of Human Platelet Antigens 1 to 6 and 15 by High-Resolution Amplicon Melting and Conventional Hybridization Probes

High-resolution melting techniques are a simple and cost-effective alternative to other closed-tube genotyping methods. Here, we genotyped human platelet antigens (HPAs) 1 to 6 and 15 by high-resolution melting methods that did not require labeled probes. Conventional melting analysis with hybridization probes (HybProbes) was also performed at each locus. HybProbe assays were performed individually, whereas amplicon melting (HPAs 1 to 5 and 16) and unlabeled probe (HPA 6) assays were duplexed when possible. At all loci for each method, both homozygous and heterozygous genotypes were easily identified. We analyzed 100 blinded clinical samples (33 amniotic fluid, 12 cultured amniocytes, and 55 blood samples) for all 7 single-nucleotide polymorphisms (SNPs) by each method. Genotype assignments could be made in 99.0% of the SNPs by high-resolution melting and in 98.7% of the SNPs with HybProbes with an overall genotype concordance of 98.8%. Errors included two sample misidentifications and six incorrect assignments that were all resolved by repeating the analysis. Advantages of high-resolution melting include rapid assay development and execution, no need for modified oligonucleotides, and similar accuracy in genotyping compared with other closed-tube melting methods.

Mutation Scanning of the RET Protooncogene Using High-Resolution Melting Analysis

Single-base pair missense mutations in exons 10, 11, 13, 14, 15, and 16 of the RET protooncogene are associated with the autosomal dominant multiple endocrine neoplasia type 2 (MEN2) syndromes: MEN2A, MEN2B, and familial medullary thyroid carcinoma. The current widely used approach for RET mutation detection is sequencing of the exons. Because RET mutations are rare and the majority are heterozygous mutations, we investigated RET mutation detection by high-resolution amplicon melting analysis. This mutation scanning technique uses a saturating double-stranded nucleic acid binding dye, LCGreen, and the high-resolution melter, HR-1, to detect heterozygous and homozygous sequence variations. Mutant genotypes are distinguished from the wild-type genotype by an altered amplicon melting curve shape or position. Samples of 26 unique RET mutations, 4 nonpathogenic polymorphisms, or the wild-type genotype were available for this study. The developed RET mutation-scanning assay differentiated RET sequence variations from the wild-type genotype by altered derivative melting curve shape or position. A blinded study of 80 samples (derived from the 35 mutant, polymorphism, or wild-type samples) demonstrated that 100% of RET sequence variations were differentiated from wild-type samples. For exons 11 and 13, the nonpathogenic polymorphisms could be distinguished from the pathogenic RET mutations. Some RET mutations could be directly genotyped by the mutation scanning assay because of unique derivative melting curve shapes. RET high-resolution amplicon melting analysis is a sensitive, closed-tube assay that can detect RET protooncogene sequence variations.

High Resolution Amplicon Melting Curve Analysis: A Sensitive and Rapid Method To Detect the Janus Kinase 2 (JAK2) Gene Mutation.

Chronic myeloproliferative disorders (CMPDs) are clonal hematopoetic stem cell disorders resulting in the increased production of cells of the myeloid lineage. The recently described G to T point mutation at nucleotide 1849 in the JAK2 gene may be a specific molecular marker to aid in the diagnosis of CMPDs, including polycythemia vera (PV), essential thrombocythemia (ET), and chronic idiopathic myelofibrosis (CIMF). Previously described methods of detection for this point mutation include sequencing, allele-specific PCR, and restriction digestion. We report here the development of a novel method to detect the JAK2 point mutation using high resolution amplicon melting curve analysis. Genomic DNA was obtained from peripheral blood samples from patients with PV, ET, CIMF, and suspected CMPD using the Qiagen DNA Blood kit (Valencia, CA). DNA was quantified using PicoGreen (Invitrogen) fluorescent dye followed by spectrophotometry and samples were normalized to a standard concentration. Real time PCR was performed on the Roche Light Cycler in the presence of the double stranded DNA binding dye LCGreen1 using primers that flank the point mutation. High Resolution Melting curve analysis was then performed on the HR-1 instrument (Idaho Technologies), and mutation status assigned by comparing the melting curve position and shape to controls of known mutation status. For comparison, the samples were also restriction digested using BsaX I and analyzed by agarose gel electrophoresis. Mutation status was confirmed by sequencing of the PCR amplicons (Lark Technologies, Houston, TX) for a subset of specimens. Twenty six patients with known CMPD as defined by the WHO criteria (2001) were included in the study. In addition, seven patients with suspected CMPD were studied. The point mutation in JAK2 was identified in 9 (100%) of 9 PV patients, 7 (58%) of 12 ET patients, 1 (50%) of 2 CIMF patients, 3 (100%) of 3 CMPD NOS patients, and 2 (29%) of 7 patients with suspected CMPD. Patients testing homozygous for the JAK2 mutation included 6 patients with PV, 1 patient with ET, and 1 patient with CMPD, NOS; all other positive patients were heterozygous for the mutation. We observed excellent correlation between the melting curve analysis and restriction digest results. High resolution melting curve analysis is a rapid, sensitive, low reagent cost method to assess for the JAK2 point mutation in cases of suspected or known CMPDs. The advantages of this technique include the ability to discriminate heterozygous from homozygous mutations, increased sensitivity as compared to sequencing, rapid turn around time, no post-amplification modification, and the ability to screen for other mutations or polymorphisms within exon 12 of the JAK2 gene.

Human malignant melanoma: detection of BRAF- and c-kit–activating mutations by high-resolution amplicon melting analysis

Activating mutations in the BRAF kinase have been reported in a large number of cases of malignant melanoma. This suggests that therapy with specific RAF kinase inhibitors may find use in treating this disease. If the response to RAF kinase inhibition is dependent on the presence of an activated BRAF protein, it will be necessary to evaluate cases of malignant melanoma for the presence or absence of BRAF mutations. High-resolution amplicon melting analysis is able to detect single base-pair changes in DNA isolated from paraffin-embedded tissue sections and obviates the need for direct DNA sequencing. Results can be available within 48 hours. In this report, we used high-resolution amplicon melting analysis to evaluate 90 cases of malignant melanoma for BRAF mutations. Of these 90 cases, 74 were metastatic melanomas, 12 were primary cutaneous melanomas, and 4 were in situ melanomas. BRAF activation mutations were found in 43 cases (48%). Forty-one of these mutations were in exon 15. The mutations in exon 15 included V600E (34 cases), V600K (6 cases), and V600R (1 case). Two activating mutations were found in exon 11, G469V and G469R. The presence or absence of a BRAF mutation in the junctional component of an invasive melanoma was maintained in the invasive component. We also evaluated these 90 cases, as well as an additional 10 cases (total of 100) for the expression of c-kit. The majority of invasive and metastatic malignant melanomas did not express c-kit, although all in situ lesions and the junctional components of invasive lesions were strongly c-kit positive. Surprisingly, 2 cases of metastatic malignant melanoma (2%) showed strong and diffuse c-kit expression and contained a c-kit-activating mutation, L576P, as detected by high-resolution amplicon melting analysis and confirmed by direct DNA sequencing. These 2 c-kit mutation-positive cases did not contain BRAF mutations. The presence of a c-kit-activating mutation in metastatic malignant melanoma suggests that a small number of melanomas may progress by a somatic mutation of the c-kit gene. The presence of BRAF- and c-kit-activating mutations in malignant melanoma suggests new approaches to treating this disease involving specific tyrosine kinase inhibitors may prove worthwhile and that mutation analysis by high-resolution melting analysis might help guide therapy.

C-KIT mutation analysis for diagnosis of gastrointestinal stromal tumors in fine needle aspiration specimens

Gastrointestinal stromal tumors (GISTs) are a specific clinicopathologic entity characterized by activating mutations in the c-KIT gene. These mutations are associated with increased immunohistochemical staining for the c-KIT protein. Because many GISTs display a definitive response to the KIT inhibitor imatinib, accurate diagnosis of these neoplasms is of great clinical importance. GISTs are the most common mesenchymal neoplasms of the digestive tract and show lineage differentiation along the lines of the interstitial cells of Cajal. The cytomorphology of GISTs has been well described, but the cytologic features are not entirely specific and immunohistochemical staining is frequently used to confirm the diagnosis. However, KIT protein positivity established by immunohistochemistry (IHC) may be unreliable in some instances because of a small number of KIT-negative GISTs and immunohistochemical KIT positivity in some non-GIST spindle cell neoplasms of the gastrointestinal tract. Antigen retrieval may be the cause of this spurious positivity. Recently, the demonstration of KIT and PDGFRA mutations has been shown to be more reliable in establishing the diagnosis of GIST than IHC. High resolution amplicon melting analysis was performed in a series of eight gastrointestinal stromal tumors sampled by fine needle aspiration (FNA). In five of eight cases, adequate material was obtained for mutational analysis. In four of these, mutations in the c-KIT gene were detected, and in the fifth case a mutation in the PDGFRA gene was detected. It appears that high resolution amplicon melting analysis can be successfully performed on material obtained by FNA and will show either KIT for PDGFR mutations in the majority of GIST FNA specimens.

Detection of c-kit–Activating Mutations in Gastrointestinal Stromal Tumors by High-Resolution Amplicon Melting Analysis

High-resolution amplicon melting analysis was used to scan for c-kit-activating mutations in exons 9, 11, 13, and 17 in 29 neoplasms diagnosed as gastrointestinal stromal tumors (GISTs). Immunohistochemically, 7 of 29 did not show strong CD 17 positivity and might represent true smooth muscle tumors or c-kit-negative GISTs. No c-kit-activating mutations were detected in the 7 CD117- cases by high-resolution amplicon melting analysis or direct DNA sequencing. Alterations in the remaining 22 CD117+ cases included 13 (59%) in exon 11, 2 (9%) in exon 9, 1 (5%) in exon 13, and none in exon 17. The genetic alterations consisted of point mutations and in-frame insertions, duplications, and deletions. In exon 11, 7 (54%) of 13 alterations have not been described previously. In 2 cases, the identical exon 11 mutation was observed in the primary tumor and a metastatic/recurrent lesion. In all cases, direct DNA sequencing confirmed that polymerase chain reaction products with an abnormal melting curve contained a mutation and products with a normal melting curve, a normal DNA sequence. High-resolution melting analysis can be used to scan DNA for potential c-kit-activating mutations and can aid in the diagnosis of GISTs.

High-resolution genotyping by amplicon melting analysis using LCGreen.

High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49:396-406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides. We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), beta-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. The six common beta-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. 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.