Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.     

A novel method based on high resolution melting (HRM) analysis for MIRU-VNTR genotyping of Mycobacterium tuberculosis

The mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method is one of the most important methods that have been used in recent years for genotyping Mycobacterium tuberculosis. Agarose gel electrophoresis and capillary electrophoresis have been used to determine the size of amplicons, however, both of these methods have shortcomings. Here, we develop and evaluate a novel method for MIRU-VNTR typing based on high resolution melting (HRM) analysis. The MIRU40 locus was selected to evaluate different real-time PCR machines and the accuracy of our method; the Roche LightCycler 480 provided greatest consistency between the T_m value and repeat number and was used in subsequent evaluations. Our method gives greater accuracy in comparison with conventional agarose gel electrophoresis (98.9% vs. 90.9%, p = 0.017), and, with the help of fitting formulae, can be used to obtain the number of MIRU tandem repeats from the T_m value. To validate our method we analyzed 12 classical MIRU loci to genotype 88 clinical isolates. The number of MIRU tandem repeats was determined accurately, quickly and conveniently.     

The detection and functions of RNA modification m6A based on m6A writers and erasers

N⁶-methyladenosine (m⁶A) is the most frequent chemical modification in eukaryotic mRNA and is known to participate in a variety of physiological processes, including cancer progression and viral infection. The reversible and dynamic m⁶A modification is installed by m⁶A methyltransferase (writer) enzymes and erased by m⁶A demethylase (eraser) enzymes. m⁶A modification recognized by m⁶A binding proteins (readers) regulates RNA processing and metabolism, leading to downstream biological effects such as promotion of stability and translation or increased degradation. The m⁶A writers and erasers determine the abundance of m⁶A modifications and play decisive roles in its distribution and function. In this review, we focused on m⁶A writers and erasers and present an overview on their known functions and enzymatic molecular mechanisms, showing how they recognize substrates and install or remove m⁶A modifications. We also summarize the current applications of m⁶A writers and erasers for m⁶A detection and highlight the merits and drawbacks of these available methods. Lastly, we describe the biological functions of m⁶A in cancers and viral infection based on research of m⁶A writers and erasers and introduce new assays for m⁶A functionality via programmable m⁶A editing tools.     

TILLING,high-resolution melting (HRM), and next-generation sequencing (NGS) techniques in plant mutation breeding

Induced mutations have been used effectively for plant improvement. Physical and chemical mutagens induce a high frequency of genome variation. Recently, developed screening methods have allowed the detection of single nucleotide polymorphisms (SNPs) and the identification of traits that are difficult to identify at the molecular level by conventional breeding. With the assistance of reverse genetic techniques, sequence variation information can be linked to traits to investigate gene function. Targeting induced local lesions in genomes (TILLING) is a high-throughput technique to identify single nucleotide mutations in a specific region of a gene of interest with a powerful detection method resulted from chemical-induced mutagenesis. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of genome size and ploidy level. However, TILLING requires laborious and time-consuming steps, and a lack of complete genome sequence information for many crop species has slowed the development of suitable TILLING targets. Another method, high-resolution melting (HRM), which has assisted TILLING in mutation detection, is faster, simpler and less expensive with non-enzymatic screening system. Currently, the sequencing of crop genomes has completely changed our vision and interpretation of genome organization and evolution. Impressive progress in next-generation sequencing (NGS) technologies has paved the way for the detection and exploitation of genetic variation in a given DNA or RNA molecule. This review discusses the applications of TILLING in combination with HRM and NGS technologies for screening of induced mutations and discovering SNPs in mutation breeding programs.     

Glucose-6-phosphate dehydrogenase (G6PD) gene mutations detection by improved high-resolution DNA melting assay

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide including southern China. G6PD gene mutations cause deficiency of the enzyme and a large spectrum of diseases. High-resolution DNA melting (HRM) assay was recently introduced as a rapid, inexpensive and effective method for genotyping. But there was a shortcoming of this method that hemizygous and homozygous genotypes were not easily distinguished from wild-types. Here we used improved HRM method for a small-scale screening of G6PD-deficient variants among people of Meizhou region. Then all amplicons were ascertained by direct DNA sequencing. These results indicated that HRM method was a major technical advance for G6PD mutations screening.     

ScreenClust: Advanced statistical software for supervised and unsupervised high resolution melting (HRM) analysis

Background: High resolution melting (HRM) is an emerging new method for interrogating and characterizing DNA samples. An important aspect of this technology is data analysis. Traditional HRM curves can be difficult to interpret and the method has been criticized for lack of statistical interrogation and arbitrary interpretation of results. Methods: Here we report the basic principles and first applications of a new statistical approach to HRM analysis addressing these concerns. Our method allows automated genotyping of unknown samples coupled with formal statistical information on the likelihood, if an unknown sample is of a known genotype (by discriminant analysis or “supervised learning”). It can also determine the assortment of alleles present (by cluster analysis or “unsupervised learning”) without a priori knowledge of the genotypes present. Conclusion: The new algorithms provide highly sensitive and specific auto-calling of genotypes from HRM data in both supervised an unsupervised analysis mode. The method is based on pure statistical interrogation of the data set with a high degree of standardization. The hypothesis-free unsupervised mode offers various possibilities for de novo HRM applications such as mutation discovery.     

Use of high-resolution melting and melting temperature-shift assays for specific detection and identification of Bacillus anthracis based on single nucleotide discrimination

Single nucleotide polymorphisms (SNPs) are important diagnostic markers for the detection and differentiation of Bacillus anthracis. High-Resolution Melting (HRM) and Melting Temperature (Tm)-shift methods are two approaches that enable SNP detection without the need for expensive labeled probes. We evaluated the potential diagnostic capability of those methods to discriminate B. anthracis from the other members of the B. cereus group. Two assays targeting B. anthracis-specific SNPs in the plcR and gyrA genes were designed for each method and used to genotype a panel of 155 Bacilli strains. All B. anthracis isolates (n = 65) were correctly and unambiguously identified. Assays also proved to be appropriate for the direct genotyping of biological samples. They could reliably detect B. anthracis in contaminated organs containing as little as 10³ CFU/ml, corresponding to a few genome equivalents per reaction. The HRM and Tm-shift applications described here represent valuable tools for specific identification of B. anthracis at reduced cost.     

Deep and accurate detection of m6A RNA modifications using miCLIP2 and m6Aboost machine learning

N6-methyladenosine (m⁶A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m⁶A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m⁶A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m⁶A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m⁶A detection. The optimized miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m⁶A detection. The analyses were calibrated with Mettl3 knockout cells to learn the characteristics of m⁶A deposition, including m⁶A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m⁶A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m⁶A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m⁶A identification.     

Roles of N6‐methyladenosine (m6A) RNA modifications in urological cancers

Epigenetics has long been a hot topic in the field of scientific research. The scope of epigenetics usually includes chromatin remodelling, DNA methylation, histone modifications, non‐coding RNAs and RNA modifications. In recent years, RNA modifications have emerged as important regulators in a variety of physiological processes and in disease progression, especially in human cancers. Among the various RNA modifications, m⁶A is the most common. The function of m⁶A modifications is mainly regulated by 3 types of proteins: m⁶A methyltransferases (writers), m⁶A demethylases (erasers) and m⁶A‐binding proteins (readers). In this review, we focus on RNA m⁶A modification and its relationship with urological cancers, particularly focusing on its roles and potential clinical applications.     

A m6A Sensing Method by Its Impact on the Stability of RNA Double Helix

N⁶‐Methyladenosine (m⁶A) is one of the most important RNA modifications in epigenetics. The development of detection method for m⁶A is limited by its abundance and structure. Although it has been previously reported that its presence has an impact on the complementary pairing of RNA, few assays have been developed using this finding. We used this discovery and designed a detection method based on Cas13a system, which has different fluorescence signals for target RNAs containing m⁶A modification and target RNAs without m⁶A modification. We verified the fact that the presence of m⁶A could cause the instability of dsRNA using the Cas13a system and provided a new direction and strategy for the development of m⁶A detection methods in the future.     

Optimizing high-resolution melting analysis for the detection of mutations of GPR30/GPER-1 in breast cancer

G protein-coupled receptor 30/G protein estrogen receptor-1 (GPR30/GPER-1) is a novel membrane receptor for estrogen whose mRNA is expressed at high levels in estrogen-dependent cells such as breast cancer cell lines. However, mutations in GRP30 related to diseases remain unreported. To detect unknown mutations in the GPR30 open reading frame (ORF) quickly, the experimental conditions for high-resolution melting (HRM) analysis were examined for PCR primers, Taq polymerases, saturation DNA binding dyes, Mg(2+) concentration, and normalized temperatures. Nine known SNPs and 13 artificial point mutations within the GPR30 ORF, as well as single nucleotide variants in DNA extracted from subjects with breast cancers were tested under the optimal experimental conditions. The combination of Expand High Fidelity(PLUS) and SYTO9 in the presence of 2.0 mM MgCl(2) produced the best separation in melting curves of mutations in all regions of the GPR30 ORF. Under these experimental conditions, the mutations were clearly detected in both heterozygotes and homozygotes. HRM analysis of GPR30 using genomic DNA from subjects with breast cancers showed a novel single nucleotide variant, 111C>T in GPR30 and 4 known SNPs. The experimental conditions determined in this study for HRM analysis are useful for high throughput assays to detect unknown mutations within the GPR30 ORF.     

N~6 -methyl-adenosine (m ~6A) in RNA: An Old Modification with A Novel Epigenetic Function

N6 -methyl-adenosine (m6A) is one of the most common and abundant modifications on RNA molecules present in eukaryotes. However, the biological significance of m6A methylation remains largely unknown. Several independent lines of evidence suggest that the dynamic regulation of m6A may have a profound impact on gene expression regulation. The m6A modification is catalyzed by an unidentified methyltransferase complex containing at least one subunit methyltransferase like 3 (METTL3). m6A modification on messenger RNAs (mRNAs) mainly occurs in the exonic regions and 3’-untranslated region (3’-UTR) as revealed by high-throughput m6A-seq. One significant advance in m6A research is the recent discovery of the first two m6A RNA demethylases fat mass and obesity-associated (FTO) gene and ALKBH5, which catalyze m6A demethylation in an a-ketoglutarate (a-KG)-and Fe2+-dependent manner. Recent studies in model organisms demonstrate that METTL3, FTO and ALKBH5 play important roles in many biological processes, ranging from development and metabolism to fertility. Moreover, perturbation of activities of these enzymes leads to the disturbed expression of thousands of genes at the cellular level, implicating a regulatory role of m6A in RNA metabolism. Given the vital roles of DNA and histone methylations in epigenetic regulation of basic life processes in mammals, the dynamic and reversible chemical m6A modification on RNA may also serve as a novel epigenetic marker of profound biological significances.     

DNA barcode ITS2 coupled with high resolution melting (HRM) analysis for taxonomic identification of Sideritis species growing in Greece

Identification of genotypes in Sideritis is complicated owing to the morphological similarity and common occurrence of natural hybridisation within Sideritis species. Species- and genotype-specific DNA markers are very useful for plant identification, breeding and preservation programs. Herein, a real-time polymerase chain reaction (PCR) of ITS2 barcode region coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on seven Sideritis species growing in Greece. The HRM assay developed in this study is a rapid and straightforward method for the identification and discrimination of the investigated Sideritis species. This assay is simple compared to other genotyping methods as it does not require DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of Sideritis species.     

Conformational analysis of the tetranucleotides m6(2)A-m6(2)A-U-m6(2)A(m6(2)A = N6-dimethyladenosine) and U-m6(2)A-U-m6(2)A and of the hybrid dA-r(U-A). A one- and two-dimensional NMR study

A 1H-NMR investigation was carried out on the tetranucleotides U-m6(2)A-U-m6(2)A and m6(2)A-m6(2)A-U-m6(2)A (m6(2) = N6-dimethyladenosine) as well as on the hybrid trinucleotide dA-r(U-A). An extensive comparison with m6(2)A-U-m6(2)A and other relevant compounds is made. Previous proton NMR studies on trinucleotides have shown that purine-pyrimidine-purine sequences prefer to adopt a mixture of states which have as a common feature that the interior pyrimidine residue bulges out, whereas the flanking purine residues stack upon each other. A stacking interaction on the 3' side of the bulge is known to have no measurable effect on the bulge population. Chemical-shift data, ribose ring conformational analysis and information from NOE experiments now show unambiguously that the moderate U(1)-m6(2)A(2) stack in U-m6(2)A-U-m6(2)A diminishes the population of bulged-out structures in favour of a regular stack. This tendency towards conformational transmission in the downstream 5'----3' direction is fully confirmed by the fact that the strong m6(2)A(1)-m6(2)A(2) stack in the tetranucleotide m6(2)A-m6(2)A-U-m6(2)A virtually precludes the formation of bulged-out structures. The conformational characteristics of dA-r(U-A) appear comparable with those of m6(2)A-U-m6(2)A, which indicates that the presence of a 2'-hydroxyl group in the first purine residue is not a necessary prerequisite for the formation of a bulge.     

Influencing factors of dsDNA dye (high-resolution) melting curves and improved genotype call based on thermodynamic considerations

High-resolution melting of dsDNA using suitable dyes is a simple and cost-effective alternative for mutation scanning. Analytical variation can result from salt and template concentration (C_T). To overcome this problem the van’t Hoff transition enthalpy ΔH_vH from dsDNA melting curves was estimated and used for robust genotype calling or mutation scanning. Model calculations show the effect of salt, C_T, and temperature resolution on (1) T_m, (2) difference plots, (3) melting peaks, and (4) calculated ΔH_vH. Using the LightCycler480, the influence of dye (ResoLight) and scanning speed was assessed. The model calculations show that only ΔH_vH is not influenced by salt and C_T. Higher amplicon enthalpy ameliorates the ability to discriminate mutations. Temperature resolution is important for peak- but not for curve-based genotyping. ResoLight increases T_m by 3.4 °C, while lowering ΔH_vH. Using a 4-bp deletion in a 200-bp amplicon as a model, the miscalling rate improved substantially, when using ΔH_vH instead of difference plots. Melting curves of duplex DNA are influenced by dye and salt and less so by duplex concentrations. As predicted from theory, ΔH_vH is a robust measure for mutation detection in two-state melting. The influence of dyes on enthalpy is of general impact for PCR assays.     

A cost-effective high-resolution melting approach using the EvaGreen dye for DNA polymorphism detection and genotyping in plants

High-resolution melting (HRM) analysis relies on the use of fluorescent dyes, such as LCGreen,ResoLight, and SYTO9, which bind in a saturated manner to the double-stranded DNAs. These dyes are expensive in use and may not be affordable when dealing with a large quantity of samples. EvaGreen is a much cheaper DNA helix intercalating dye and has been used in quantitative real-time polymerase chain reaction (PCR) and post-PCR DNA melt curve analysis. Here we report on the development of an EvaGreen-based HRM analysis and its performance, in comparison with the popular LCGreen-based HRM analysis, in detection of DNA polymorphism in plants. We found that various polymorphisms ranged from single nucleotide polymorphisms (SNPs) to Indels were equally detected by using EvaGreen- or LCGreen-based HRM. EvaGreen dye was sensitive enough in discovery of SNPs in fivefold pooled samples.Using this economical dye we successfully identified multiple novel mutant alleles of Gln1-3 gene,which produces a cytosolic glutamine synthetase isoenzyme (GS1), in a maize ethyl methanesulfonate (EMS)-mutagenized library, and genotyped rice mapping populations with SNP markers. The current results suggest that EvaGreen is a promising dye for HRM analysis for its ease to use and cost effectiveness.