A Cost-effective High-resolution Melting Approach using the EvaGreen Dye for DNA Polymorphism Detection and Genotyping in Plants

2010 ◽  
Vol 52 (12) ◽  
pp. 1036-1042 ◽  
Author(s):  
Yi-Dan Li ◽  
Zhi-Zhan Chu ◽  
Xiang-Guo Liu ◽  
Hai-Chun Jing ◽  
Yao-Guang Liu ◽  
...  
Keyword(s):  
High Resolution ◽  
Dna Polymorphism ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Polymorphism Detection

scholarly journals Genotyping of GATA4 Gene Variant (G296S) in Malaysian Congenital Heart Disease Subjects by Real-Time PCR High Resolution Melting Analysis

2013 ◽  
Vol 32 (2) ◽  
pp. 152-157
Author(s):  
Nora Fawzi ◽  
Ramachandran Vasudevan ◽  
Patimah Ismail ◽  
Mazeni Alwi ◽  
Ahmad Fazli Abdul Aziz ◽  
...  
Keyword(s):  
Congenital Heart Disease ◽  
Heart Disease ◽  
High Resolution ◽  
Congenital Heart ◽  
High Resolution Melting ◽  
New Technology ◽  
Cost Effective ◽  
Study Cohort ◽  
Hrm Analysis ◽  
Melting Analysis

Summary Background: Congenital heart disease (CHD) is the most common birth defect; however, the underlying etiology is unrecognized in the majority of cases. GATA-binding protein 4 (GATA4), a cardiac transcription factor gene, has a crucial role in the cardiogenesis process; hence, a number of heterozygote sequence variations were identified as a cause of CHD. G296S heterozygote variant is the most frequently reported GATA4 gene sequence alteration. This study aims to investigate the role of G296S variant of the GATA4 gene in Malaysian CHD subjects. Methods: We have investigated 86 Malaysian CHD subjects with cardiac septation defects for the presence of the GATA4 gene heterozygote variant (G296S) by the new technology of high resolution melting (HRM) analysis. Results: Genotyping of G296S (c.886G>A) by HRM analysis shows that all the sample genotypes were of the wild GG type genotype and the heterozygote mutant GA genotype was totally absent from this study cohort. Conclusions: The results of our study showed that the G296S variant of the GATA4 gene was not associated with the development of CHD in Malaysian subjects. The use of HRM analysis proved a cost-effective, high-throughput, specific and sensitive genotyping technique which eliminates the need for unnecessary sequencing.

scholarly journals High-Resolution Melting Is a Sensitive, Cost-Effective, Time-Saving Technique for BRAF V600E Detection in Thyroid FNAB Washing Liquid: A Prospective Cohort Study

2015 ◽  
Vol 4 (2) ◽  
pp. 73-81 ◽  
Author(s):  
Marco Marino ◽  
Maria Laura Monzani ◽  
Giulia Brigante ◽  
Katia Cioni ◽  
Bruno Madeo ◽  
...  
Keyword(s):  
Cohort Study ◽  
High Resolution ◽  
Molecular Analysis ◽  
Prospective Cohort Study ◽  
Prospective Cohort ◽  
Large Scale ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Braf V600e Mutation ◽  
Braf V600e

Objective: The diagnostic accuracy of thyroid fine needle aspiration biopsy (FNAB) can be improved by the combination of cytological and molecular analysis. In this study, washing liquids of FNAB (wFNAB) were tested for the BRAF V600E mutation, using the sensitive and cost-effective technique called high-resolution melting (HRM). The aim was to demonstrate the feasibility of BRAF analysis in wFNAB and its diagnostic utility, combined with cytology. Design: Prospective cohort study. Methods: 481 patients, corresponding to 648 FNAB samples, were subjected to both cytological (on cells smeared onto a glass slide) and molecular analysis (on fluids obtained washing the FNAB needle with 1 ml of saline) of the same aspiration. BRAF V600E analysis was performed by HRM after methodological validation for application to wFNAB (technique sensitivity: 5.4%). Results: The cytological results of the FNAB were: 136 (21%) nondiagnostic (THY1); 415 (64%) benign (THY2); 80 (12.4%) indeterminate (THY3); 9 (1.4%) suspicious for malignancy (THY4); 8 (1.2%) diagnostic of malignancy (THY5). The BRAF V600E mutation was found in 5 THY2, 2 THY3, 6 THY4 and 6 THY5 samples. Papillary carcinoma diagnosis was histologically confirmed in all BRAF+ thyroidectomized patients. BRAF combined with cytology improved the diagnostic value compared to cytology alone in a subgroup of 74 operated patients. Conclusions: HRM was demonstrated to be a feasible method for BRAF analysis in wFNAB. Thanks to its sensitivity and cost-effectiveness, it might be routinely used on a large scale in clinical practice. In perspective, standby wFNAB samples could be analyzed a posteriori in case of indeterminate cytology and/or suspicious findings on ultrasound.

scholarly journals High Resolution Melting and Insertion Site-Based Polymorphism Markers for Wheat Variability Analysis and Candidate Genes Selection at Drought and Heat MQTL Loci

Agronomy ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1294 ◽  
Author(s):  
Rosa Mérida-García ◽  
Sergio Gálvez ◽  
Etienne Paux ◽  
Gabriel Dorado ◽  
Laura Pascual ◽  
...  
Keyword(s):  
Quantitative Trait Loci ◽  
High Resolution ◽  
Quantitative Trait ◽  
High Resolution Melting ◽  
Insertion Site ◽  
Cost Effective ◽  
Wheat Breeding ◽  
Reference Sequence ◽  
Detection Techniques ◽  
Trait Loci

The practical use of molecular markers is facilitated by cost-effective detection techniques. In this work, wheat insertion site-based polymorphisms (ISBP) markers were set up for genotyping using high-resolution melting analysis (HRM). Polymorphic HRM-ISBP assays were developed for wheat chromosomes 4A and 3B and used for wheat variability assessment. The marker sequences were mapped against the wheat genome reference sequence, targeting interesting genes. Those genes were located within or in proximity to previously described quantitative trait loci (QTL) or meta-quantitative trait loci (MQTL) for drought and heat stress tolerance, and also yield and yield related traits. Eighteen of the markers used tagged drought related genes and, interestingly, eight of the genes were differentially expressed under different abiotic stress conditions. These results confirmed HRM as a cost-effective and efficient tool for wheat breeding programs.

scholarly journals High-Resolution Melting-Based Quantitative Analysis of RASSF1 Methylation in Breast Cancer

Medicina ◽  
2013 ◽  
Vol 49 (2) ◽  
pp. 14 ◽  
Author(s):  
Kristina Stuopelytė ◽  
Kristina Daniūnaitė ◽  
Aida Laurinavičienė ◽  
Valerijus Ostapenko ◽  
Sonata Jarmalaitė
Keyword(s):  
Breast Cancer ◽  
Polymerase Chain Reaction ◽  
High Resolution ◽  
Quantitative Methods ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Chain Reaction ◽  
Specific Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Breast Tissues

Background and Objective. Breast cancer is the leading cause of death from cancer among women worldwide. The aberrant promoter methylation of tumor suppressor genes is a typical epigenetic alteration for breast cancer and can be detected in early carcinogenesis. High-throughput and cost-effective methods are needed for the early and sensitive detection of epigenetic changes in clinical material. The main purpose of our study was to optimize a high-resolution melting (HRM) assay for the reliable and quantitative assessment of RASSF1 gene methylation, which is considered one of the earliest epigenetic alterations in breast cancer. Material and Methods. A total of 76 breast carcinomas and 10 noncancerous breast tissues were studied by means of HRM and compared with the results obtained by means of quantitative methylation-specific polymerase chain reaction (QMSP) and methylation-specific polymerase chain reaction (MSP). Results. Both quantitative methods, HRM and QMSP, showed a similar specificity and sensitivity for the detection of RASSF1 methylation in breast cancer (about 80% and 70%, respectively). In breast cancer, the mean methylation intensity of RASSF1 was 42.5% and 48.6% according to HRM and QMSP, respectively. Both methods detected low levels of methylation (less than 5%) in noncancerous breast tissues. In comparison with quantitative methods, MSP showed a lower sensitivity (70%), but a higher specificity (80%) for the detection of RASSF1 methylation in breast cancer. Conclusions. HRM is as a simple, cost-effective method for the reliable high-throughput quantification of DNA methylation in clinical material.

scholarly journals A simple and cost-effective approach for technical validation of next generation methylation sequencing data

2019 ◽  
Author(s):  
Ali Javadmanesh ◽  
Afsaneh Mojtabanezhad Shariatpanahi ◽  
Ehsan Shams Davodly ◽  
Marjan Azghandi ◽  
Maryam Yassi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
High Resolution ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Next Generation ◽  
Sequencing Data ◽  
Cost Effective Approach ◽  
Technical Validation ◽  
R Programming ◽  
And Control

Abstract Background DNA methylation is a fundamental epigenetic process that, in most cases, modulates genetic expression levels. Changes in DNA methylation, either hypo- or hypermethylation, have a key role in many biological processes and several human diseases such as cancer. In the current study, we offered an approach to validate the next generation methylation sequencing data.Methods Genomic DNA was extracted from target and control samples (6 in each group), followed by bisulfite conversion. Next generation methylation sequencing and methylation sensitive high-resolution melting assay were carried out. The primers for methylation sequencing validation were designed by R programming language.Results In the current study, two groups, case and control, were discriminated based on methylation sequencing results and the real time PCR-based results were in accordance with the next generation methylation sequencing.Discussion Methylation sensitive high-resolution melting validation assay is a simple and cost-effective method, which confirmed next generation methylation sequencing results.

scholarly journals High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Lucas B. Harrison ◽  
Nancy D. Hanson
Keyword(s):  
Escherichia Coli ◽  
Principal Component Analysis ◽  
High Resolution ◽  
High Resolution Melting ◽  
Principal Component ◽  
Cost Effective ◽  
Sequence Type ◽  
Content Type ◽  
E Coli ◽  
Sequence Types

ABSTRACT Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification.

Rapid and cost effective detection of small mutations in the DMD gene by high resolution melting curve analysis

2009 ◽  
Vol 19 (6) ◽  
pp. 383-390 ◽  
Author(s):  
Rowida Almomani ◽  
Nienke van der Stoep ◽  
Egbert Bakker ◽  
Johan T. den Dunnen ◽  
Martijn H. Breuning ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Cost Effective ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
High Resolution Melting Curve ◽  
Dmd Gene

scholarly journals Traceability of PDO Olive Oil “Terra di Bari” Using High Resolution Melting

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Cinzia Montemurro ◽  
Monica Marilena Miazzi ◽  
Antonella Pasqualone ◽  
Valentina Fanelli ◽  
Wilma Sabetta ◽  
...  
Keyword(s):  
High Resolution ◽  
Olive Oil ◽  
High Resolution Melting ◽  
Virgin Olive Oil ◽  
Cost Effective ◽  
Extra Virgin Olive Oil ◽  
Hrm Analysis ◽  
Olive Cultivars ◽  
Pcr Products ◽  
Set Up

The aim of the research was to verify the applicability of microsatellite (SSR) markers in High Resolution Melting (HRM) analysis for the identification of the olive cultivars used in the “Terra di Bari” PDO extra virgin olive oil. A panel of nine cultivars, widespread in Apulia region, was tested with seventeen SSR primer pairs and the PCR products were at first analysed with a Genetic Analyzer automatic sequencer. An identification key was obtained for the nine cultivars, which showed an unambiguous discrimination among the varieties constituting the “Terra di Bari” PDO extra virgin olive oil: Cima di Bitonto, Coratina, and Ogliarola. Subsequently, an SSR based method was set up with the DCA18 marker, coupled with HRM analysis for the distinction of the Terra di Bari olive oil from non-Terra di Bari olive oil using different mixtures. Thus, this analysis enabled the distinction and identification of the PDO mixtures. Hence, this assay provided a flexible, cost-effective, and closed-tube microsatellite genotyping method, well suited to varietal identification and authentication analysis in olive oil.

scholarly journals Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies

2017 ◽  
Vol 63 (10) ◽  
pp. 1605-1613 ◽  
Author(s):  
Ioannis Ladas ◽  
Mariana Fitarelli-Kiehl ◽  
Chen Song ◽  
Viktor A Adalsteinsson ◽  
Heather A Parsons ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Digital Pcr ◽  
Clinical Samples ◽  
Wild Type ◽  
Liquid Biopsies ◽  
Targeted Resequencing ◽  
Double Stranded Dna ◽  
Closed Tube

Abstract BACKGROUND The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing. METHODS We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA. RESULTS Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10–20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing. CONCLUSIONS Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing.

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