DNA Melting Curve Analysis Research Articles

Association of the rs1042522 SNP with prostate cancer risk: a study of cancer tissues, primary tumor cultures, and serum samples from a Spanish Caucasian population.

Prostate cancer (PCa) is a leading cause of cancer-related deaths in European men, emphasizing the urgent need for effective risk assessment strategies. The TP53 gene, a tumor suppressor gene frequently mutated in cancer, commonly harbors the rs1042522 single nucleotide polymorphism (SNP), known as the P72R SNP, which may influence PCa susceptibility. This study investigated the prevalence of the P72R SNP in European Caucasian PCa samples and its association with PCa risk. Genotyping was conducted on 12 hormone-naïve aggressive PCa cultures (hnPCs) from untreated patients (Gleason ≥8), 11 radical prostatectomies (RP), and 94 serum samples using DNA Sanger sequencing and melting curve analysis. Comparative analysis utilized data from the GnomAD database's European Caucasian non-cancer population. Our results demonstrate a significantly higher frequency of the P72R SNP in PCa samples and serums compared to the general European non-cancer population. A robust and statistically significant association (p < 0.0001) between the SNP and prostate cancer risk was identified, with an odds ratio of 7.937 (95% CI 5.37-11.00). Notably, the G allele (R72) showed a pronounced prevalence in high Gleason score (≥8) patients, although statistical significance was not reached. These results highlight a potential association with undifferentiated and malignant PCa lesions. The compelling association between the P72R SNP and prostate cancer risk underscores the potential utility of this marker for the early identification of patients at risk of aggressive metastatic prostate cancer. This insight could empower further research to intervene at an early stage by offering enhanced opportunities for timely and targeted interventions.

Elucidation of the interaction of apocarotenoids with calf thymus DNA by biophysical techniques and in vitro study in MCF-7 cells to explore their potential in cancer therapy.

DNA is one of the targets of cancer-therapeutic small molecules. Cisplatin, a DNA intercalator, is one of the first-line drugs in the cancer chemo regimen which comes with health-compromising side effects during chemotherapy. The synergistic effect of natural molecules with cisplatin can help to potentiate its anti-cancer efficacy and decrease its negative effect on health. Here, we report the interaction of cisplatin with calf thymus-DNA (ct-DNA) in combination with natural molecules like apocarotenoids which are reported for their therapeutic properties. The combinatorial effect of apocarotenoids on ct-DNA was explored through various biophysical techniques such as UV-Visible spectroscopy, circular dichroism studies, DNA melt curve analysis, viscosity measurements, and an in vitro study in MCF-7 cells by cell cycle analysis. UV-Visible spectroscopy studies suggest apocarotenoids and their combination shows a non-intercalative mode of binding. Circular dichroism analysis showed no major changes in DNA form during the interaction of DNA with apocarotenoids and their respective combinations with cisplatin, which is suggestive of the groove-binding mode of apocarotenoids. DNA melt curve analysis showed a decrease in the intensity of the fluorescence for apocarotenoids with cisplatin which indicates the possibility of DNA interaction through groove binding. Viscosity studies suggested a groove binding mode of interaction of ct-DNA with apocarotenoids and their combination as there was minimal change in the viscosity measurements. The in vitro analysis exhibits that the apocarotenoids and their combination have a considerable effect on DNA synthesis. This study provides a better perspective on the possible mode of interaction between ct-DNA and natural molecules along with cisplatin.

Development of DNA Melt Curve Analysis for the Identification of Lepidopteran Pests in Almonds and Pistachios.

Simple SummaryAlmonds and pistachios are fed upon by a diverse assemblage of lepidopteran insects, several of which are economically important pests. Unfortunately, identification of these pests can be difficult, as specimens are frequently damaged during collection, occur in traps with non-target species, and are morphologically similar up to their third instar. Here, we present a quantitative PCR based melt curve analysis for simple, rapid, and accurate identification of six lepidopteran pests of almonds and pistachios: navel orangeworm, peach twig borer, oriental fruit moth, obliquebanded leafroller, raisin moth, and Indian meal moth. We demonstrate that the dissociation or the “melt” temperature(s) of a 658 bp section of cytochrome c oxidase subunit 1 provides unambiguous species level identification of these six species and is reproducible in field specimens collected following conventional orchard practices. The melt curve’s simplicity allows it to be performed in any basic molecular biology laboratory with a quantitative PCR.Almonds and pistachios are fed upon by a diverse assemblage of lepidopteran insects, several of which are economically important pests. Unfortunately, identification of these pests can be difficult, as specimens are frequently damaged during collection, occur in traps with non-target species, and are morphologically similar up to their third instar. Here, we present a quantitative PCR based melt curve analysis for simple, rapid, and accurate identification of six lepidopteran pests of almonds and pistachios: navel orangeworm (Amyelois transitella), peach twig borer (Anarsia lineatella), oriental fruit moth (Grapholita molesta), obliquebanded leafroller (Choristoneura rosaceana), raisin moth (Cadra figulilella), and Indian meal moth (Plodia interpunctella). In this approach, the dissociation (melt) temperature(s) of a 658 bp section of cytochrome c oxidase subunit 1 was determined using quantitative PCR (qPCR). Within these six species, the distribution and the number of melt peak temperatures provide an unambiguous species level identification that is reproducible when unsheared DNA can be extracted. The test is robust across a variety of sampling approaches including insects removed from sticky card traps, museum specimens, and samples that were left in the field for up to 7 days. The melt curve’s simplicity allows it to be performed in any basic molecular biology laboratory with a quantitative PCR.

Application of machine learning algorithm and modified high resolution DNA melting curve analysis for molecular subtyping of Salmonella isolates from various epidemiological backgrounds in northern Thailand.

Food poisoning from consumption of food contaminated with non-typhoidal Salmonella spp. is a global problem. A modified high resolution DNA melting curve analysis (m-HRMa) was introduced to provide effective discrimination among closely related HRM curves of amplicons generated from selected Salmonella genome sequences enabled Salmonella spp. to be classified into discrete clusters. Combination of m-HRMa with serogroup identification (ms-HRMa) helped improve assignment of Salmonella spp. into clusters. In addition, a machine learning (dynamic time warping) algorithm (DTW) was employed to provide a simple and rapid protocol for clustering analysis as well as to create phylogeny tree of Salmonella strains (n = 40) collected from home, farms and slaughter houses in northern Thailand. Applications of DTW and ms-HRMa clustering analyses were capable of generating molecular signatures of the Salmonella isolates, resulting in 25ms-HRM and 28 DTW clusters compared to 14 clusters from a standard HRM analysis, and the combination of both analyses permitted molecular subtyping of each Salmonella isolate. Results from DTW and ms-HRMa cluster analyses were in good agreement with that obtained from enterobacterial repetitive intergenic consensus sequence PCR clustering. While conventional serotyping of Clusters 1 and 2 revealed six different Salmonella serotypes, the majority being S. Weltevraden, the new Salmonella subtyping protocol identified five S. Weltevraden subtypes with S.Weltevreden subtype DTW4-M1 being predominant. Based on knowledge of the sources of Salmonella subtypes, transmission of S. Weltevraden in northern Thailand was likely to be farm-to-farm through contaminated chicken stool. In conclusion, the rapid, robust and specific Salmonella subtyping developed in the study can be performed in a local setting, enabling swift control and preventive measures to be initiated against potential epidemics of salmonellosis.

Live-cell PCR and one-step purification streamline DNA engineering.

In vivo DNA engineering such as recombineering (recombination-mediated genetic engineering) and DNA gap repair typically involve growing Escherichia coli (E coli) containing plasmids, followed by plasmid DNA extraction and purification prior to downstream PCR-mediated DNA modifications and DNA sequencing. We previously demonstrated that crude cell lysates could be used for some limited downstream DNA applications. Here, we show how live E coli cell PCR and one-step LiCl-isopropanol purification can streamline DNA engineering. In DNA gap repair, live-cell PCR allowed the convenient elimination of clones containing background plasmids prior to DNA sequencing. Live-cell PCR also enabled the generation of specific DNA sequences for DNA engineering up to 11 kilo base pairs in length and with up to 80 base pair terminal non-homology. Using gel electrophoresis and DNA melting curve analysis, we showed that LiCl-isopropanol DNA precipitation removed primers and small, nonspecific PCR products from live-cell PCR products in only ~10-minutes. DNA sequencing of purified products yielded Phred quality scores values of ~55%. These data indicate that live-cell PCR and LiCl-isopropanol DNA precipitation are ideal to prepare DNA for sequencing and other downstream DNA applications, and might therefore accelerate high-throughput DNA engineering pipelines.

Comparison of cattle BoLA-DRB3 typing by PCR-RFLP, direct sequencing, and high-resolution DNA melting curve analysis.

Major histocompatibility complex (MHC) represents an important genetic marker for manipulation to improve the health and productivity of cattle. It is closely associated with numerous disease susceptibilities and immune responses. Bovine MHC, also called bovine leukocyte antigen (BoLA), is considered as a suitable marker for genetic diversity studies. In cattle, most of the polymorphisms are located in exon 2 of BoLA-DRB3, which encodes the peptide-binding cleft. In this study, the polymorphism of the BoLA-DRB3.2 gene in Holstein's calves was studied using high resolution melting curve analysis (HRM). Observed HRM results were compared to PCR-RFLP and direct sequencing techniques. Eight different HRM and seven different RFLP profiles were identified among the population studied. By comparing to sequencing data, HRM could completely discriminate all genotypes (eight profiles), while the RFLP failed to distinguish between the genotypes *1101/*1001 and *1104/*1501. According to the results, the HRM analysis method gave more accurate results than RFLP by differentiating between the BoLA-DRB3.2 genotypes. Due to the Co-dominant nature of the MHC alleles, HRM technique could be used for investigating the polymorphisms of genotypes and their associations with immune responses.

Heat transfer time determination based on DNA melting curve analysis

The determination of the physical properties of fluids—such as the thermal characteristics, which include heat transfer time (Δt)—is becoming more challenging as system sizes shrink to micro- and nanometer scales. Hence, knowledge of these properties is crucial for the operation of devices requiring precise temperature (T) control, such as polymerase chain reactions, melting curve analysis (MCA), and differential scanning fluorimetry. In this paper, we introduced a flow-through microfluidic system to analyze thermal properties such as Δt among samples and the sidewall of a silicon chip using microscopic image analysis. We performed a spatial MCA with double-stranded deoxyribonucleic acid (dsDNA) and EvaGreen intercalator, using a flow-through microfluidic chip, and achieved a T gradient of ≈ 2.23 K mm−1. We calculated the mean value of Δt as ≈ 33.9 ms from a melting temperature (TM) location shift along the microchannel for a variable flow rate. Our system had a T resolution of ≈ 1.2 mK pixel−1 to distinguish different dsDNA molecules—based on the TM location within the chip—providing an option to use it as a high-throughput device for rapid DNA or protein analysis.

Asymmetric mutant-enriched polymerase chain reaction and quantitative DNA melting analysis of KRAS mutation in colorectal cancer

Identification of mutant genes in tumor tissues and blood plasma (solid and liquid biopsy samples, respectively) is a necessity for individualized treatment of cancer patients. Here we report the use of a novel mutant-enriched PCR - quantitative DNA melting curve analysis (mePCR-qDMA) with TaqMan probes. The TaqMan probes served as blocking agents during PCR and as hybridization probes during DNA melting curve analyses. The end-point measurement of melt peaks areas by PeakFit software, a nonlinear iterative curve-fitting program, permitted quantification of the mutant/wild-type allele ratios. Approximately 6% and 0.1% of mutant KRAS allele in an excess of wild-type allele is detected with the standard and mePCR-qDMA processes, respectively. The application of the approach was tested for detecting the KRAS codon 12/13 mutation in paired tumor and blood plasma samples from 20 colorectal cancer patients. KRAS mutants were detected in 7 and 18 FFPE tumor samples, and in 3 and 7 plasma samples by the standard and mePCR-qDMA process, respectively. The results were confirmed by Sanger sequencing. This simple, rapid, cost-effective, and quantitative method carried out in a closed-tube format could be applied for the clinical analyses of other cancer genes.

Removal of artifact bias from qPCR results using DNA melting curve analysis.

Quantitative PCR (qPCR) allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, analyses using validated Sybr Green I-based assays regularly amplify both the correct product and an artifact. Amplification of more than 1 product can be recognized when melting curve analysis is performed after the qPCR. Currently, such reactions need to be excluded from further analysis because the quantification result is considered meaningless. However, when the fraction of the fluorescence associated with the correct product can be determined, the quantitative result of the qPCR analysis can be corrected. The main assumptions of this correction model are: 1) the melting peak of the correct product can be identified, 2) the PCR efficiencies of all amplified products are similar, 3) the relative size of the melting peaks reflects the relative concentrations of the products, and 4) the relative concentrations do not change as the reaction reaches plateau. These assumptions were validated in a series of model experiments. The results show that the quantitative results can be corrected. Implementation of a correction for the presence of artifact amplification in the analysis of qPCR data leads to more reliable quantitative results in qPCR experiments.-Ruijter, J. M., Ruiz-Villalba, A., van den Hoff, A. J. J., Gunst, Q. D., Wittwer, C. T., van den Hoff, M. J. B. Removal of artifact bias from qPCR results using DNA melting curve analysis.

Variant Intestinal-Cell Kinase in Juvenile Myoclonic Epilepsy

BackgroundIn juvenile myoclonic epilepsy, data are limited on the genetic basis of networks promoting convulsions with diffuse polyspikes on electroencephalography (EEG) and the subtle microscopic brain dysplasia called microdysgenesis.MethodsUsing Sanger sequencing, we sequenced the exomes of six members of a large family affected with juvenile myoclonic epilepsy and confirmed cosegregation in all 37 family members. We screened an additional 310 patients with this disorder for variants on DNA melting-curve analysis and targeted real-time DNA sequencing of the gene encoding intestinal-cell kinase (ICK). We calculated Bayesian logarithm of the odds (LOD) scores for cosegregating variants, odds ratios in case–control associations, and allele frequencies in the Genome Aggregation Database. We performed functional tests of the effects of variants on mitosis, apoptosis, and radial neuroblast migration in vitro and conducted video-EEG studies in mice lacking a copy of Ick.ResultsA variant, K305T (c.914A→C), cosegregated with epilepsy or polyspikes on EEG in 12 members of the family affected with juvenile myoclonic epilepsy. We identified 21 pathogenic ICK variants in 22 of 310 additional patients (7%). Four strongly linked variants (K220E, K305T, A615T, and R632X) impaired mitosis, cell-cycle exit, and radial neuroblast migration while promoting apoptosis. Tonic–clonic convulsions and polyspikes on EEG resembling seizures in human juvenile myoclonic epilepsy occurred more often in knockout heterozygous mice than in wild-type mice (P=0.02) during light sleep with isoflurane anesthesia.ConclusionsOur data provide evidence that heterozygous variants in ICK caused juvenile myoclonic epilepsy in 7% of the patients included in our analysis. Variant ICK affects cell processes that help explain microdysgenesis and polyspike networks observed on EEG in juvenile myoclonic epilepsy. (Funded by the National Institutes of Health and others.)

DNA methylation assay using droplet-based DNA melting curve analysis.

DNA methylation is an epigenetic regulation of gene expression, which has drawn great attention in biomedical research due to its association with various diseases. A robust, inexpensive platform to detect and quantify the methylation status in a specific genomic region is necessary. In this study, an on-chip analytical technique of cytosine methylation with droplets in a microchannel is proposed. Genomic DNA samples are encapsulated into a series of droplets and transported through a detection region, where a stable temperature gradient is created. As the temperature is elevated from 60 °C to 85 °C, the DNA samples denature and the associated fluorescence signals decay, with the relationship being acquired as the melting curve. The droplets serve as discrete reactors for conducting DNA melting curve analysis in the liquid phase, thereby eliminating the need for immobilization of reagents. Due to a high heating rate and greater enhanced thermal stability, this microchip allows larger melting temperature differences for the samples at different percentages of methylated DNA. It has an enhanced discrimination ability and lower volume consumption, compared to the commercial qPCR machine. This chip enables quantification of the methylation levels of the pluripotent stem cell factor Oct-4 in its distal enhancer (DE) region, with a designed probe after bisulfite treatment and asymmetric PCR.

Sub-7-second genotyping of single-nucleotide polymorphism by high-resolution melting curve analysis on a thermal digital microfluidic device.

We developed a thermal digital microfluidic (T-DMF) device enabling ultrafast DNA melting curve analysis (MCA). Within 7 seconds, the T-DMF device succeeded in differentiating a melting point difference down to 1.6 °C with a variation of 0.3 °C in a tiny droplet sample (1.2 μL), which was 300 times faster and with 20 times less sample spending than the standard MCA (35 minutes, 25 μL) run in a commercial qPCR machine. Such a performance makes it possible for a rapid discrimination of single-nucleotide mutation relevant to prompt clinical decision-making. Also, aided by electronic intelligent control, the T-DMF device facilitates sample handling and pipelining in an automatic serial manner. An optimized oval-shaped thermal electrode is introduced to achieve high thermal uniformity. A device-sealing technique averts sample contamination and permits uninterrupted chemical/biological reactions. Simple fabrication using a single chromium layer fulfills both the thermal and typical transport electrode requirements. Capable of thermally modulating DNA samples with ultrafast MCA, this T-DMF device has the potential for a wide variety of life science analyses, especially for disease diagnosis and prognosis.

Rapid detection of immunoglobulin heavy chain gene rearrangement by PCR and melting curve analysis using combined FR2 and FR3 primers

BackgroundImmunoglobulin heavy chain (IgH) gene rearrangement test is a standard tool in diagnosing B-cell lymphoma. The BIOMED-2 multiplex PCR protocol has become the most commonly used laboratory method for detecting clonal IgH gene rearrangement. However, post-PCR procedure requires manual transfer of PCR product for analysis and is time-consuming. A novel strategy using LightCycler to continuously monitor fluorescence during melting curve analysis (MCA) can overcome these shortcomings. The previous studies published on this method were all restricted to FR3 primers of BIOMED-2.MethodsReal-time PCR and subsequent MCA were performed on 71 clinical DNA samples from formalin-fixed, paraffin-embedded tissues, including 40 with B-cell non-Hodgkin lymphomas and 31 with reactive lymphoid hyperplasia. We optimized the current method using FR3 primers and applied FR2 primers for the first time into MCA to detect IgH gene rearrangement. Polyacrylamide gel electrophoresis and capillary gel electrophoresis were also performed on all lymphoma samples with the identical FR2 primers.ResultsMCA of combined FR2 and FR3 primer sets yielded the sensitivity and the specificity equal to 70 % (28/40) and 100 % (31/31), respectively. Addition of FR2 primers increased the sensitivity by 12.5 % (5/40) comparing to FR3 primers alone. MCA was slightly more sensitive than polyacrylamide gel electrophoresis and comparable to capillary gel electrophoresis to detect clonal IgH gene rearrangement.ConclusionsCombined PCR and DNA melting curve analysis in a closed system can reduce cross-contamination risk. This method can test 96 samples simultaneously within 90 min and therefore, it is high-throughput and faster. PCR-MCA in the LightCycler system has potential for evaluating monoclonal IgH gene rearrangement in a clinical environment.

Genotyping of velvet antlers for identification of country of origin using mitochondrial DNA and fluorescence melting curve analysis with locked nucleic acid probes

Velvet antlers are used medicinally in Asia and possess various therapeutic effects. Prices are set according to the country of origin, which is unidentifiable to the naked eye, and therefore counterfeiting is prevalent. Additionally, antlers of the Canadian elk, which can generate chronic wasting disease, are prevalently smuggled and distributed in the market. Thus, a method for identifying the country of origin of velvet antlers was developed, using polymorphisms in mitochondrial DNA, fluorescence melting curve analysis and analysis of locked nucleic acids (LNA). This combined method is capable of identifying five genotypes of velvet antlers in a single experiment using two probes. It also has advantages in multiplexing, simplicity and efficiency in genotyping, when compared to real-time PCR or microarrays. The developed method can be used to improve identification rates in the velvet antler market and, by extension, research based on polymorphisms in DNA sequences.

Charging YOYO-1 on Capillary Wall for Online DNA Intercalation and Integrating This Approach with Multiplex PCR and Bare Narrow Capillary–Hydrodynamic Chromatography for Online DNA Analysis

Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary–hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions.

On-chip magnetic bead-based DNA melting curve analysis using a magnetoresistive sensor

We present real-time measurements of DNA melting curves in a chip-based system that detects the amount of surface-bound magnetic beads using magnetoresistive magnetic field sensors. The sensors detect the difference between the amount of beads bound to the top and bottom sensor branches of the differential sensor geometry. The sensor surfaces are functionalized with wild type (WT) and mutant type (MT) capture probes, differing by a single base insertion (a single nucleotide polymorphism, SNP). Complementary biotinylated targets in suspension couple streptavidin magnetic beads to the sensor surface. The beads are magnetized by the field arising from the bias current passed through the sensors. We demonstrate the first on-chip measurements of the melting of DNA hybrids upon a ramping of the temperature. This overcomes the limitation of using a single washing condition at constant temperature. Moreover, we demonstrate that a single sensor bridge can be used to genotype a SNP.

Rapid genotyping of beak and feather disease virus using high-resolution DNA melt curve analysis

Beak and feather disease virus (BFDV) is a significant pathogen both for wild and captive psittacine birds globally. Genotypic differentiation of BFDV isolates is crucial to establish effective control strategies for the conservation of endangered species and epidemiological investigations of disease outbreaks. The technique developed in this study is a simple, rapid and inexpensive genotyping method for BFDV using PCR and subsequent high-resolution melt (HRM) curve analysis. This was achieved using PCR amplification of the conserved Rep gene in the presence of a fluorescent DNA intercalating dye (SYTO9). HRM curve analysis of the resultant amplicon could readily differentiate between reference strain (92-SR14) and 18 other BFDV isolates used in this study. Analysis of the nucleotide sequences of the amplicon from each isolate revealed that each melt curve profile was related to a unique DNA sequence. The potential of the PCR–HRM curve analysis to differentiate inter-host genetic variation among critically endangered orange-bellied parrots, lorikeets and cockatoos was also evaluated. Phylogenetic tree topology based on partial Rep gene sequences used in this study showed that BFDV Rep gene sequence patterns were correlated with the results of HRM curve analysis. The results presented in this study indicate that this technique could be used in both clinical research and differentiation of BFDV isolates in a fraction of time without further nucleotide sequencing and provides a novel approach for the genetic screening of BFDV in clinical virology laboratories.

Application of Different Molecular Techniques for Characterization of Catalase‐Positive Cocci Isolated from Sucuk

This study was carried out for the characterization and discrimination of the indigenous Gram positive, catalase-positive cocci (GCC) population in sucuk, a traditional Turkish dry-fermented sausage. Sucuk samples, produced by the traditional method without starter culture were collected from 8 local producers in Kayseri/Turkey and a total of 116 GCC isolates were identified by using different molecular techniques. Two different molecular fingerprinting methods; namely, randomly amplified polymorphic DNA-PCR (RAPD-PCR) and repetitive extragenic palindrome-PCR (rep-PCR), were used for the clustering of isolates and identification at species level was carried out by full length sequencing of 16S rDNA. Combining the results obtained from molecular fingerprinting and 16S rDNA sequencing showed that the dominant GCC species isolated from the sucuk samples was Staphylococcus saprophyticus followed by Staphylococcus succinus and Staphylococcus equorum belonging to the Staphylococcus genus. Real-time PCR DNA melting curve analysis and high-resolution melting (HRM) analysis targeting the V1 + V3 regions of 16S rDNA were also applied for the discrimination of isolates belonging to different species. It was observed statistically different Tm values and species-specific HRM profiles for all except 2 species (S. saprophyticus and Staphylococcus xylosus) that have high 16S rDNA sequence similarity. The combination of rep-PCR and/or PCR-RAPD with 16S rRNA gene sequencing was an efficient approach for the characterization and identification of the GCC population in spontaneously fermented sucuk. On the other hand, intercalating dye assays were found to be a simple and very promising technique for the differentiation of the GCC population at species level.

Rapid molecular diagnosis of the Gilbert's syndrome-associated exon 1 mutation within the UGT1A1 gene

Gilbert's syndrome is suspected in patients with unconjugated hyperbilirubinemia caused by decreased activity of the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene in the absence of abnormal liver function and hemolysis. The major genetic variants underlying Gilbert's syndrome are TATA-box repeats of the promoter region and exon 1 G211A of the coding region, particularly in Asians. The efficacy of DNA melting curve analysis, however, has not been established for the G211A mutation. For rapid and accurate molecular diagnosis of Gilbert's syndrome, DNA melting curve analysis was evaluated for its genotyping capability not only for TATA-box repeats of the UGT1A1 promoter, but also for G211A of UGT1A1 exon 1. TA repeats within the TATA-box sequence and the exon 1 G211A mutation of the UGT1A1 gene were analyzed by DNA melting curve analysis. To evaluate the assay reliability, direct sequencing or polyacrylamide gel electrophoresis was used as a comparative method. All homozygous and heterozygous polymorphisms of A(TA)7TAA within the TATA-box allele and of exon 1 G211A mutants of the UGT1A1 gene were successfully identified with DNA melting curve analysis. DNA melting curve analysis is, therefore, an effective molecular method for the rapid diagnosis of Gilbert's syndrome, as it detects not only TATA-box polymorphisms but also the exon 1 G211A mutation located within the UGT1A1 gene.

High‐resolution DNA melt‐curve analysis for cost‐effective mass screening of pairwise species interactions

Ecological studies of pairwise interactions are constrained by the methods available for rapid species identification of the interacting organisms. The resolution of data required to characterize species interaction networks at multiple spatio-temporal scales can be intensive, and therefore laborious and costly to collect. We explore the utility of high-resolution DNA melt-curve analysis (HRM) as a rapid species identification method. An approach was developed to identify organisms at the pairwise interaction level, with particular application to cryptic species interactions that are traditionally difficult to study. Here, we selected a challenging application; to identify the presence/absence of pathogenic fungi (Sporothrix inflata, Ophiostoma nigrocarpum and Ophiostoma galeiforme) transported by bark beetle vectors (Hylastes ater and Hylurgus ligniperda). The technique was able to distinguish between different species of DNA within a single, pooled sample. In test applications, HRM was effective in the mass screening and identification of pathogenic fungal species carried by many individual bark beetle vectors (n=455 beetles screened) across large geographic scales. For two of the fungal species, there was no difference in the frequency of association with either of their vectors, but for the third fungal species there was a shift in vector-pathogen associations across locations. This technique allows rapid, mass screening and characterization of species interactions at a fraction of the time and cost of traditional methods. It is anticipated that this method can be readily applied to explore other cryptic species interactions, or other studies requiring rapid generation of large data sets and/or high-throughput efficiency.