Discovering Rare Variants by Use of Melting Temperature Shifts Seen in Melting Curve Analysis

Detection of V1016G mutation is important for identifying the mechanism of synthetic pyrethroid resistance in Aedes aegypti population. The previous method has described an allele specific polymerase chain reaction (AS-PCR) using conventional PCR to detect the mutation. Although the method has great differentiating power and reproducibility, faster and more sensitive genotyping method is essential to accurately detect the mutation. This study evaluate the used of SYBR® Green real-time PCR and melting curve analysis (MCA) to identify the V1016G mutation. The collection of homozygous 1016G, heterozygous, and wild type (1016 V) mosquitoes DNA genome was extracted using genomic DNA mini kit. The SsoAdvanced™ Universal SYBR® Green Supermix was used to identify alleles by real-time PCR followed melting curve analysis of the amplicons. Melting curve analysis produced reproducible results for the loci tested. The melting temperature was reached at 78.5 oC for homozygous 1016G mosquito and at 86 oC for wild type mosquito. Meanwhile, the heterozigous mosquito revealed two peaks of melting temperature at both 78.5 oC and 86 oC. These easily interpretable and distinguishable melting curve results were consistent with AS-PCR results obtained for the same alleles. The described MCA application for screening V1016G mutation is fast and widely accessible also could be implemented under field conditions

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Web-based LinRegPCR: application for the visualization and analysis of (RT)-qPCR amplification and melting data

BMC Bioinformatics ◽

10.1186/s12859-021-04306-1 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Andreas Untergasser ◽

Jan M. Ruijter ◽

Vladimir Benes ◽

Maurice J. B. van den Hoff

Keyword(s):

Melting Temperature ◽

Melting Curve ◽

Curve Analysis ◽

Melting Curve Analysis ◽

Amplification Product ◽

Web Based ◽

Amplification Curve ◽

Qpcr Data ◽

Melting Curves ◽

The Individual

Abstract Background The analyses of amplification and melting curves have been shown to provide valuable information on the quality of the individual reactions in quantitative PCR (qPCR) experiments and to result in more reliable and reproducible quantitative results. Implementation The main steps in the amplification curve analysis are (1) a unique baseline subtraction, not using the ground phase cycles, (2) PCR efficiency determination from the exponential phase of the individual reactions, (3) setting a common quantification threshold and (4) calculation of the efficiency-corrected target quantity with the common threshold, efficiency per assay and Cq per reaction. The melting curve analysis encompasses smoothing of the observed fluorescence data, normalization to remove product-independent fluorescence loss, peak calling and assessment of the correct peak by comparing its melting temperature with the known melting temperature of the intended amplification product. Results The LinRegPCR web application provides visualization and analysis of a single qPCR run. The user interface displays the analysis results on the amplification curve analysis and melting curve analysis in tables and graphs in which deviant reactions are highlighted. The annotated results in the tables can be exported for calculation of gene-expression ratios, fold-change between experimental conditions and further statistical analysis. Web-based LinRegPCR addresses two types of users, wet-lab scientists analyzing the amplification and melting curves of their own qPCR experiments and bioinformaticians creating pipelines for analysis of series of qPCR experiments by splitting its functionality into a stand-alone back-end RDML (Real-time PCR Data Markup Language) Python library and several companion applications for data visualization, analysis and interactive access. The use of the RDML data standard enables machine independent storage and exchange of qPCR data and the RDML-Tools assist with the import of qPCR data from the files exported by the qPCR instrument. Conclusions The combined implementation of these analyses in the newly developed web-based LinRegPCR (https://www.gear-genomics.com/rdml-tools/) is platform independent and much faster than the original Windows-based versions of the LinRegPCR program. Moreover, web-based LinRegPCR includes a novel statistical outlier detection and the combination of amplification and melting curve analyses allows direct validation of the amplification product and reporting of reactions that amplify artefacts.

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Genotyping UGT1A1(TA)n polymorphism rare variants by high resolution melting curve analysis

Clinica Chimica Acta ◽

10.1016/j.cca.2010.11.029 ◽

2011 ◽

Vol 412 (5-6) ◽

pp. 489-490 ◽

Cited By ~ 5

Author(s):

Barbara Ostanek ◽

Danijela Furlan ◽

Borut Bratanič

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Rare Variants ◽

Melting Curve ◽

Curve Analysis ◽

Melting Curve Analysis ◽

High Resolution Melting Curve

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Rapid Differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi Sensu Stricto by LightCycler Fluorescence Melting Curve Analysis of a PCR Product of the recA Gene

Journal of Clinical Microbiology ◽

10.1128/jcm.38.7.2756-2759.2000 ◽

2000 ◽

Vol 38 (7) ◽

pp. 2756-2759 ◽

Cited By ~ 47

Author(s):

Johanna Pietilä ◽

Qiushui He ◽

Jarmo Oksi ◽

Matti K. Viljanen

Keyword(s):

Borrelia Burgdorferi ◽

Melting Temperature ◽

Melting Curve ◽

Sensu Stricto ◽

Curve Analysis ◽

Reca Gene ◽

Melting Curve Analysis ◽

Pcr Product ◽

Pcr Products ◽

Borrelia Burgdorferi Sensu Stricto

To differentiate the Borrelia burgdorferi sensu lato genospecies, LightCycler real-time PCR was used for the fluorescence (SYBR Green I) melting curve analysis of borrelial recAgene PCR products. The specific melting temperature analyzed is a function of the GC/AT ratio, length, and nucleotide sequence of the amplified product. A total of 32 DNA samples were tested. Of them three were isolated from B. burgdorferi reference strains and 16 were isolated from B. burgdorferi strains cultured fromIxodes ricinus ticks; 13 were directly isolated from nine human biopsy specimens and four I. ricinus tick midguts. The melting temperature of B. garinii was 2°C lower than that of B. burgdorferi sensu stricto and B. afzelii. Melting curve analysis offers a rapid alternative for identification and detection of B. burgdorferi sensu lato genospecies.

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Genotyping of Entamoeba histolytica by Real-Time Polymerase Chain Reaction with Sybr Green I and Melting Curve Analysis

Bangladesh Journal of Veterinary Medicine ◽

10.3329/bjvm.v4i1.1526 ◽

1970 ◽

Vol 4 (1) ◽

pp. 53-60

Author(s):

SMM Rahman ◽

R Haque ◽

S Roy ◽

MMH Mondal

Keyword(s):

Real Time ◽

Melting Temperature ◽

Entamoeba Histolytica ◽

Real Time Pcr ◽

Melting Curve ◽

Curve Analysis ◽

Sybr Green ◽

Sybr Green I ◽

Melting Curve Analysis ◽

Intestinal Amoebiasis

In this study, for the detection of distinct genotype of E. histolytica of human, a nested Real-Time PCR amplification of SREHP gene using SYBR Green I and melting curve analysis was done. A total of 60 specimens (stool and liver aspirate specimens), which were found Entamoeba histolytica positive by E. histolytica specific ELISA and ssrRNA gene PCR, were selected and the experiment was conducted during the period of July 2003 to June 2004. After melting curve analysis of amplified PCR products from these isolates of stool and liver aspirate specimens, 5 genotypes were found belonging to the melting temperatures 84Â°C, 83Â°C, 82Â°C, 81Â°C and 79Â°C. All these 5 genotypes were present in intestinal amoebiasis patients and when the genotypes from intestinal amoebiasis patients were compared with the genotypes of amoebic liver amoebiasis patients, the genotype 84Â°C melting temperature was found to be absent in amoebic liver amoebiasis patients. For both the cases of intestinal and amoebic liver amoebiasis patients the genotype belonging to 83Â°C melting temperature was more prevalent than the other genotypes which suggest that this genotype is more responsible for the development of amoebiasis. In comparison to conventional PCR method where we found 23 different banding patterns, the Real-Time PCR and melting curve analysis method was found to be more reliable for the detection of distinct genotypes of E. histolytica because with this method we found only 5 genotypes. In conclusion, this Real-Time PCR using SYBR Green I and melting curve analysis for the genotyping of E. histolytica, excludes the need of post PCR manipulations and would be helpful for the rapid detection and screening of E. histolytica genotypes among endemic population and also for the epidemiological study. Key words: Entamoeba histolytica, genotype, diarrhoea, Real-Time PCR, melting curve analysis doi:10.3329/bjvm.v4i1.1526 Bangl. J. Vet. Med. (2006). 4 (1): 53-60

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Melting Curve Analysis of Aptachains: Adenosine Detection with Internal Calibration

Biosensors ◽

10.3390/bios11040112 ◽

2021 ◽

Vol 11 (4) ◽

pp. 112

Author(s):

Chenze Lu ◽

Christine Saint-Pierre ◽

Didier Gasparutto ◽

Yoann Roupioz ◽

Corinne Ravelet ◽

...

Keyword(s):

Small Molecules ◽

Melting Temperature ◽

Melting Curve ◽

Temperature Shift ◽

Curve Analysis ◽

Detection Methods ◽

Melting Curve Analysis ◽

High Melting ◽

Internal Calibration ◽

High Melting Temperature

Small molecules are ubiquitous in nature and their detection is relevant in various domains. However, due to their size, sensitive and selective probes are difficult to select and the detection methods are generally indirect. In this study, we introduced the use of melting curve analysis of aptachains based on split-aptamers for the detection of adenosine. Aptamers, short oligonucleotides, are known to be particularly efficient probes compared to antibodies thanks to their advantageous probe/target size ratio. Aptachains are formed from dimers with dangling ends followed by the split-aptamer binding triggered by the presence of the target. The high melting temperature of the dimers served as a calibration for the detection/quantification of the target based on the height and/or temperature shift of the aptachain melting peak.

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