Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis

This protocol permits the simultaneous mutation scanning and genotyping of PCR products by high-resolution DNA melting analysis. This is achieved using asymmetric PCR performed in the presence of a saturating fluorescent DNA dye and unlabeled oligonucleotide probes. Fluorescent melting curves of both PCR amplicons and amplicon-probe duplexes are analyzed. The shape of the PCR amplicon melting transition reveals the presence of heterozygotes, whereas specific genotyping is enabled by melting of the unlabeled probe-amplicon duplex. Unbiased hierarchal clustering of melting transitions automatically groups different sequence variants; this allows common variants to be easily recognized and genotyped. This technique may be used in both laboratory research and clinical settings to study single-nucleotide polymorphisms and small insertions and deletions, and to diagnose associated genetic disorders. High-resolution melting analysis accomplishes simultaneous gene scanning and mutation genotyping in a fraction of the time required when using traditional methods, while maintaining a closed-tube environment. The PCR requires <30 min (capillaries) or 1.5 h (96- or 384-well plates) and melting acquisition takes 1-2 min per capillary or 5 min per plate.     

Allele-specific extension allows base-pair neutral homozygotes to be discriminated by high-resolution melting of small amplicons

Not all single-nucleotide polymorphisms (SNPs) can be determined using high-resolution melting (HRM) of small amplicons, especially class 3 and 4 SNPs. This is due mainly to the small shift in the melting temperature (Tm) between two types of homozygote. Choosing rs1869458 (a class 4 SNP) as a sample, we developed a modified small amplicon HRM assay. An allele-specific extension (ASE) primer, which ended at an SNP site and matched only one of the alleles, was added to the reaction as well as additional thermal steps for ASE. Following asymmetric polymerase chain reaction and melting curve analysis, heterozygotes were easily identified. Two types of homozygote were also distinguishable, indicating that extension primers 11 to 13 bases in length worked efficiently in an allele-specific way. Modification of the limiting amplification primer with locked nucleic acid increased the Tm difference between extension and amplification peaks and facilitated subsequent genotyping. In addition, 194 human genomic DNA samples were genotyped with the developed assay and by direct sequencing, with the different methods providing identical genotyping results. In conclusion, ASE-HRM is a simple, inexpensive, closed-tube genotyping method that can be used to examine all types of SNP.     

High-Throughput Genotyping by Coupling Adapter-Ligation Mediated Allele-Specific Amplification with Microplate Array Parallel Gel Electrophoresis

Recently, we have developed a method of adapter-ligation mediated allele-specific amplification (ALM-ASA) for simultaneously typing multiple single nucleotide polymorphisms (SNPs) at a low cost. We usually use agarose gel-electrophoresis for analyzing PCR products. As the processes of sampling and PCR can be carried out at a format of 96-well or 384-well, the throughput-bottleneck of whole process of ALM-ASA is only the agarose gel-electrophoresis. Here we improved the typing throughput of ALM-ASA by using a microplate array parallel gel electrophoresis (MAPGE) system, with which 96 amplicons can be detected at a time. By coupling with multiplexed preamplification, seven SNPs distributed on four different human genes (IL1A (549C>T), 1L1B (794C>T and 5277C>T), IL10 (2940G>A, 3203C>T, and 3430C>A), and TNFA (1431G>A)) were successfully typed. The optimization of allele-specific primers in ALM-ASA was performed by the software of “SNiPdesigner” which was designed especially for ALM-ASA. We also demonstrated that the specificity of ALM-ASA assay for SNP typing is superior to that of amplification refractory mutation system (ARMS).     

Studies of type I collagen (<Emphasis Type="Italic">COL1A1</Emphasis>) α1 chain in patients with osteogenesis imperfecta

Nucleotide sequences of exon 51, adjacent intron areas, and regulatory region of the α1 chain of type I collagen (COL1A1) gene were analyzed in 41 patients with osteogenesis imperfecta (OI) from 33 families and their 68 relatives residing at Bashkortostan Republic (BR). Six mutations (four nonsense mutations c.967G>T (p.Gly323X), c.1081C>T (p.Arg361X), c.1243C>T (p.Arg415X), and c.2869C>T (p.Gln957X)) in patients of the Russian origin and two frameshift mutations (c.579delT (p.Gly194ValfsX71), and c.2444delG (p.Gly815AlafsX293)) in patients with OI of Tatar ethnicity as well as 14 single nucleotide polymorphisms in the COL1A1 gene were revealed. Mutations c.967G>T (p.Gly323X) and three alterations in the nucleotide sequence c.544-24C>T, c.643-36delT, and c.957 + 10insA were described for the first time.     

Applications of the method of high resolution melting analysis for diagnosis of Leber's disease and the three primary mutation spectrum of LHON in the Han Chinese population

Current screening methods, such as single strand conformational polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC) and direct DNA sequencing that are used for detecting mutation in Leber's hereditary optic neuropathy (LHON) subjects are time consuming and costly. Here we tested high-resolution melt (HRM) analysis for mtDNA primary mutations in LHON patients. In this study, we applied the high resolution melting (HRM) technology to screen mtDNA primary mutations in 50 LHON patients from their peripheral blood. In order to evaluate the reliability of this technique, we compared the results obtained by HRM and direct mtDNA sequencing. We also investigated the spectrum of three most common mtDNA mutations implicated in LHON in the Han Chinese population. The results showed HRM analysis differentiated all of the mtDNA primary mutations and identified 4 additional mtDNA mutations from 50 patients in the blind study. The prevalence of three primary mutations were 11778G>A (87.9%), 14484T>C (6.5%) and 3460G>A (1.7%) in the Han Chinese population. In conclusion, HRM analysis is a rapid, reliable, and low-cost tool for detecting mtDNA primary mutations and has practical applications in molecular genetics.     

应用HRM技术对CYP2C19*2和CYP2C19*3进行双重SNP分型

氯吡格雷是一种广泛用于预防静脉血栓形成的抗血小板药物。研究表明, 携带有CYP2C19基因功能缺失型等位基因CYP2C19*2、CYP2C19*3的病人, 其体内代谢氯吡格雷成为其活性形式的能力降低, 导致氯吡格雷抑制血小板聚集功能减弱。文章旨在建立一种利用高分辨率熔解曲线分析(High-resolution melting curve analysis,HRM)技术在闭合单管中同时对CYP2C19*2、CYP2C19*3两个多态性位点进行简便、准确分型的方法。本实验针对两个SNP位点分别设计特异性的HRM引物, 并在两个位点引物的5′端分别加上富含AT和GC的序列, 保证两个位点的扩增产物熔解峰无重叠。利用HRM技术, 快速、灵敏地对64例随机DNA样本的CYP2C19*2 、CYP2C19*3两个多态性位点进行了基因分型, 且HRM方法的分型结果与测序验证结果完全一致。因此, 利用HRM技术可以实现在闭合单管中简便、准确地对CYP2C19*2 、CYP2C19*3两个多态性位点同时进行基因分型。该方法有望应用于临床, 指导氯吡格雷的个体化用药。     

Scanning of mutations in short amplicons: Optimization of DNA melting method

The high resolution melting analysis (HRMA) is a new highly efficient method for genotyping and mutation scanning. HRMA is conducted immediately after PCR in closed-tube format, which enables the high throughput of the method. However, the closed-tube format makes HRMA dependent on the conditions of PCR and, thus, limits its capabilities. The open-tube format, which we have already developed (postamplification shortening of amplicons and optimized composition of ion medium), is applicable to the scanning of mutations of the K-RAS oncogene in tumor tissue and formalin-fixed paraffin-embedded samples. It is found that the open-tube format of DNA melting significantly increases the sensitivity of finding mutant alleles when using instruments both with and without an HRMA module. The higher sensitivity of the DNA melting compared to “Sanger” sequencing allows one to decrease the number of false-negative results of the mutation test, which is highly important for some forms of cancer.     

High resolution melting analysis of almond SNPs derived from ESTs

High resolution melting curve (HRM) is a recent advance for the detection of SNPs. The technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between samples. It has been applied to the analysis and scan of mutations in the genes causing human diseases. In plant species, the use of this approach is limited. We applied HRM analysis to almond SNP discovery and genotyping based on the predicted SNP information derived from the almond and peach EST database. Putative SNPs were screened from almond and peach EST contigs by HRM analysis against 25 almond cultivars. All 4 classes of SNPs, INDELs and microsatellites were discriminated, and the HRM profiles of 17 amplicons were established. The PCR amplicons containing single, double and multiple SNPs produced distinctive HRM profiles. Additionally, different genotypes of INDEL and microsatellite variations were also characterised by HRM analysis. By sequencing the PCR products, 100 SNPs were validated/revealed in the HRM amplicons and their flanking regions. The results showed that the average frequency of SNPs was 1:114 bp in the genic regions, and transition to transversion ratio was 1.16:1. Rare allele frequencies of the SNPs varied from 0.02 to 0.5, and the polymorphic information contents of the SNPs were from 0.04 to 0.53 at an average of 0.31. HRM has been demonstrated to be a fast, low cost, and efficient approach for SNP discovery and genotyping, in particular, for species without much genomic information such as almond.     

High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities

Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes “Jawetz” and “Heyl”, Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.     

High-resolution melting analysis to discriminate between the SARS-CoV-2 Omicron variants BA.1 and BA.2

High-resolution melting (HRM) analysis was conducted to discriminate between SARS-CoV-2 Omicron variant BA.1 (B.1.1.529.1) and subvariant BA.2 (B.1.1.529.2). We performed two-step PCR consisting of the first PCR and the second nested PCR to prepare the amplicon for HRM analysis, which detected G339D, N440K, G446S and D796Y variations in the SARS-CoV-2 spike protein. The melting temperatures (Tms) of the amplicons from the cDNA of the Omicron variant BA.1 and subvariant BA.2 receptor binding domain (RBD) in spike protein were the same: 75.2 °C (G339D variation) and 73.4 °C (D796Y variation). These Tms were distinct from those of SARS-CoV-2 isolate Wuhan-Hu-1, and were specific to the Omicron variant. In HRM analyses that detected the N440K and G446S variations, the Tms of amplicons from the cDNA of the Omicron variant BA.1 and subvariant BA.2 RBDs were 73.0 °C (N440K and G446S variations) and 73.5 °C (G446S variation). This difference indicates that the SARS-CoV-2 Omicron variants BA.1 and BA.2 can be clearly discriminated. Our study demonstrates the usefulness of HRM analysis after two-step PCR for the discrimination of SARS-CoV-2 variants.     

A novel mutation screening system for Ehlers-Danlos Syndrome, vascular type by high-resolution melting curve analysis in combination with small amplicon genotyping using genomic DNA

Ehlers-Danlos syndrome, vascular type (vEDS) (MIM #130050) is an autosomal dominant disorder caused by type III procollagen gene (COL3A1) mutations. Most COL3A1 mutations are detected by using total RNA from patient-derived fibroblasts, which requires an invasive skin biopsy. High-resolution melting curve analysis (hrMCA) has recently been developed as a post-PCR mutation scanning method which enables simple, rapid, cost-effective, and highly sensitive mutation screening of large genes. We established a hrMCA method to screen for COL3A1 mutations using genomic DNA. PCR primers pairs for COL3A1 (52 amplicons) were designed to cover all coding regions of the 52 exons, including the splicing sites. We used 15 DNA samples (8 validation samples and 7 samples of clinically suspected vEDS patients) in this study. The eight known COL3A1 mutations in validation samples were all successfully detected by the hrMCA. In addition, we identified five novel COL3A1 mutations, including one deletion (c.2187delA) and one nonsense mutation (c.2992C>T) that could not be determined by the conventional total RNA method. Furthermore, we established a small amplicon genotyping (SAG) method for detecting three high frequency coding-region SNPs (rs1800255:G>A, rs1801184:T>C, and rs2271683:A>G) in COL3A1 to differentiate mutations before sequencing. The use of hrMCA in combination with SAG from genomic DNA enables rapid detection of COL3A1 mutations with high efficiency and specificity. A better understanding of the genotype–phenotype correlation in COL3A1 using this method will lead to improve in diagnosis and treatment.     

Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR

SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye and is included in many commercially available kits at undisclosed concentrations. Binding of SG to double-stranded DNA is non-specific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis because the melting temperature (T_m) of the specific amplicon is analogous to the detection of an electrophoretic band. When using SG for real-time PCR multiplex reactions, discrimination of amplicons should be possible, provided the T_m values are sufficiently different. Real-time multiplex assays for Vibrio cholerae and Legionella pneumophila using commercially available kits and in-house SG mastermixes have highlighted variability in performance characteristics, in particular the detection of only a single product as assessed by T_m analysis but multiple products as assessed by agarose gel electrophoresis. The detected T_m corresponds to the amplicon with the higher G+C% and larger size, suggesting preferential binding of SG during PCR and resulting in the failure to detect multiple amplicons in multiplex reactions when the amount of SG present is limiting. This has implications for the design and routine application of diagnostic real-time PCR assays employing SG.     

Unique spectrum of SPAST variants in Estonian HSP patients: presence of benign missense changes but lack of exonic rearrangements

Background

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous disorder that can be an autosomal-dominant, autosomal-recessive, or X-linked disease. The most common autosomal-dominant form of the disease derives from mutations in the SPAST gene.

Methods

The aim of this study was to analyze 49 patients diagnosed with HSP from the Estonian population for sequence variants of the SPAST gene and to describe the associated phenotypes. Healthy control individuals (n = 100) with no family history of HSP were also analyzed. All patient samples were screened using denaturing high performance liquid chromatography (DHPLC) and multiplex ligation-dependent probe amplification (MLPA) assay. Samples with abnormal DHPLC and MLPA profiles were sequenced, with the same regions sequenced in control samples.

Results

Sequence variants of SPAST were identified in 19/49 HSP patients (38.8%), twelve among them had pathogenic mutations. Within the latter group there was one sporadic case. Eight patients had pure, and four - complex HSP. The twelve variants were identified: seven pathogenic (c.1174-1G>C, c.1185delA, c.1276C>T, c.1352_1356delGAGAA, c.1378C>A, c.1518_1519insTC, c.1841_1842insA) and five non-pathogenic (c.131C>T, c.484G>A, c.685A>G, c.1245+202delG, c.1245+215G>C). Only 2 of these mutations had previously been described (c.131C>T, c.1245+202delG). Three mutations, c.1174-1G>C, c.1276 C>T, c.1378C>A, showed intrafamilial segregation.

Conclusion

This study identified new variants of the SPAST gene which included benign missense variants and short insertions/deletions. No large rearrangements were found. Based on these data, 7 new pathogenic variants of HSP are associated with clinical phenotypes.     

Association of vascular endothelial growth factor gene polymorphisms with osteoporotic vertebral compression fractures in postmenopausal women

Vascular endothelial growth factor (VEGF) is involved in bone formation through its role in angiogenesis. VEGF is also known to promote the healing of fractures. Thus, we determined whether or not VEGF ?2578C>A, ?1154G>A, ?634G>C, and 936C>T polymorphisms and haplotypes are associated with osteoporotic vertebral compression fractures (OVCF) in postmenopausal Korean women. The study subjects consisted of 82 patients with osteoporotic vertebral compression fractures and 117 control postmenopausal Korean women. PCR-RFLP and real-time PCR were used to analyze the VEGF polymorphisms. Homocysteine levels were also measured to determine whether or not polymorphisms of the VEGFgene affect homocysteine/folate metabolism. The AA genotype of the ?2578C>A polymorphism was significantly different between the stroke and control groups; no significant differences in the ?1154G>A, ?634G>C, and 936C>T genotype frequencies existed. However, the A-G-G-C haplotype had a tendency to be associated with OVCF in postmenopausal Korean women. Associations between the VEGF ?2578C>A polymorphism and homocysteine levels were also noted. In summary, these results suggest that the VEGF ?2578C>A polymorphisms and VEGF haplotypes may play an important role in the etiology of OVCF in postmenopausal Korean women.     

DNA melting analysis: application of the "open tube" format for detection of mutant KRAS

High-resolution melting (HRM) analysis is a very effective method for genotyping and mutation scanning that is usually performed just after PCR amplification (the “closed tube” format). Though simple and convenient, the closed tube format makes the HRM dependent on the PCR mix, not generally optimal for DNA melting analysis. Here, the “open tube” format, namely the post-PCR optimization procedure (amplicon shortening and solution chemistry modification), is proposed. As a result, mutation scanning of short amplicons becomes feasible on a standard real-time PCR instrument (not primarily designed for HRM) using SYBR Green I. This approach has allowed us to considerably enhance the sensitivity of detecting mutant KRAS using both low- and high-resolution systems (the Bio-Rad iQ5–SYBR Green I and Bio-Rad CFX96–EvaGreen, respectively). The open tube format, though more laborious than the closed tube one, can be used in situations when maximal sensitivity of the method is needed. It also permits standardization of DNA melting experiments and the introduction of instruments of a “lower level” into the range of those suitable for mutation scanning.     

High-Resolution Melting of 12S rRNA and Cytochrome b DNA Sequences for Discrimination of Species within Distinct European Animal Families

The cheap and easy identification of species is necessary within multiple fields of molecular biology. The use of high-resolution melting (HRM) of DNA provides a fast closed-tube method for analysis of the sequence composition of the mitochondrial genes 12S rRNA and cytochrome b. We investigated the potential use of HRM for species identification within eleven different animal groups commonly found in Europe by animal-group-specific DNA amplification followed by DNA melting. Influence factors as DNA amount, additional single base alterations, and the existence of mixed samples were taken into consideration. Visual inspection combined with mathematical evaluation of the curve shapes did resolve nearly all species within an animal group. The assay can therefore not only be used for identification of animal groups and mixture analysis but also for species identification within the respective groups. The use of a universal 12S rRNA system additionally revealed a possible approach for species discrimination, mostly by exclusion. The use of the HRM assay showed to be a reliable, fast, and cheap method for species discrimination within a broad range of different animal species and can be used in a flexible “modular” manner depending on the question to be solved.     

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 analysis for bird sexing: a successful approach to molecular sex identification using different biological samples

High‐resolution melting (HRM) analysis is a very attractive and flexible advanced post‐PCR method with high sensitivity/specificity for simple, fast and cost‐effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real‐time PCR systems, along with improved saturating DNA‐binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sex‐specific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed‐tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real‐time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing.     

Mutational screening of <Emphasis Type="BoldItalic">PKD2</Emphasis> gene in the north Indian polycystic kidney disease patients revealed 28 genetic variations

Polycystic kidney disease (PKD) is a systemic disorder which adds majority of renal patients to end stage renal disease. Autosomal dominant polycystic kidney disease (ADPKD) is more prevalent and leading cause of dialysis and kidney transplant. Linkage analysis revealed some closely linked loci, two of which are identified as PKD1, PKD2 and an unidentified locus to ADPKD. This study was performed using PCR and automated DNA sequencing in 84 cases and 80 controls to test potential candidature of PKD2 as underlying cause of PKD by in silico and statistical analyses. Two associated symptoms, hypertension (19%) and liver cyst (31%) have major contribution to PKD. Gender-based analysis revealed that familial female patients (27%) and familial male patients (33%) are more hypertensive. Liver cyst, the second major contributing symptom presented by large percentage of sporadic males (46%). Genetic screening of all 15 exons of PKD2 revealed eight pathogenic (c.854_854delG, c.915C>A, c.973C>T, c.1050_1050delC, c.1604_1604delT, c.1790T>C, c.2182_2183delAG, c.2224C>T) and eight likely pathogenic (g.11732A>G, c.646T>C, c.1354A>G, g.39212G>C, c.1789C>A, c.1849C>A, c.2164G>T, c.2494A>G) DNA sequence variants. In our study, 27.38% (23/84) cases shown pathogenic / likely pathogenic variants in PKD2 gene. Some regions of PKD2 prone for genetic variation suggested to be linked with disease pathogenesis. This noticeable hot spot regions hold higher frequency (50%) of pathogenic / likely pathogenic genetic variants constituting single nucleotide variants than large deletion and insertion that actually represents only 41.08% of coding sequence of PKD2. Statistically significant association for IVS3-22AA genotype was observed with PKD, while association of IVS4+62C>T was found insignificant.     

The use of high-resolution melting analysis for genotyping Symbiodinium strains: a sensitive and fast approach

High-resolution melting (HRM) analysis is a closed-tube, rapid and sensitive technique able to detect DNA variations. It relies on the fluorescence melting curves that are obtained from the transition of double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA) as a result of temperature increase. In this study, we evaluated the effectiveness of HRM as a tool to rapidly and precisely genotype monotypic Symbiodinium populations using the internal transcribed spacer, region 2, ribosomal DNA (ITS2 rDNA). For this, Symbiodinium denaturing gradient gel electrophoresis (DGGE) profiles, where gel bands were excised and sequenced, were compared to HRM genotypes. Results showed that twenty cultures were correctly genotyped in <2 h using HRM analysis with a percentage of confidence >90%. Limitations of the technique were also investigated. Unlike other techniques used for genotyping Symbiodinium, such as DGGE and other fingerprint profiles, HRM is a technique of great advantage for field coral reef ecologists and physiologists as no expertise in advanced molecular methods is required.