DNA melting analysis

Wittwer, Carl T.; Hemmert, Andrew C.; Kent, Jana O.; Rejali, Nick A.

doi:10.1016/j.mam.2024.101268

Cited by 4 publications

(5 citation statements)

References 90 publications

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“…In the qPCR melting curve analysis, SYBR green fluorescent dye was used to label double-stranded DNA. As the temperature increased, the DNA separated (melted), causing a decrease in florescence ( Wittwer et al., 2024 ). The melting temperature was specific to the B. dorsalis DNA sequences.…”

Section: Resultsmentioning

confidence: 99%

A simple PCR-based quick detection of the economically important oriental fruit fly, Bactrocera dorsalis (Hendel) from India

Arya,

Narayana,

Sinha

et al. 2024

Front. Plant Sci.

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The oriental fruit fly, Bactrocera dorsalis (Hendel), is a significant economic and quarantine pest due to its polyphagous nature. The accurate identification of B. dorsalis is challenging at the egg, maggot, and pupal stages, due to lack of distinct morphological characters and its similarity to other fruit flies. Adult identification requires specialized taxonomist. Existing identification methods are laborious, time consuming, and expensive. Rapid and precise identification is crucial for timely management. By analyzing the variations in the mitochondrial cytochrome oxidase-1 gene sequence (Insect barcoding gene), we developed a species-specific primer (SSP), DorFP1/DorRP1, for accurate identification of B. dorsalis. The optimal annealing temperature for the SSP was determined to be 66°C, with no cross-amplification or primer-dimer formation observed. The SSP was validated with B. dorsalis specimens from various locations in northern and eastern India and tested for cross-specificity with six other economically significant fruit fly species in India. The primer specificity was further confirmed by the analysis of critical threshold (Ct) value from a qPCR assay. Sensitivity analysis showed the primer could detect template DNA concentrations as low as 1 pg/µl, though sensitivity decreased at lower concentrations. Sequencing of the SSP-amplified product revealed over >99% similarity with existing B. dorsalis sequences in the NCBI GenBank. The developed SSP reliably identifies B. dorsalis across all developmental stages and sexes. This assay is expected to significantly impact pest identification, phytosanitary measures, and eradication programs for B. dorsalis.

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Section: Resultsmentioning

confidence: 99%

A simple PCR-based quick detection of the economically important oriental fruit fly, Bactrocera dorsalis (Hendel) from India

Arya,

Narayana,

Sinha

et al. 2024

Front. Plant Sci.

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“…Melting is faster than gel preparation and electrophoresis. Moreover, data analysis is not biased; it is performed automatically and provides statistical metrics [19]. Considering that the accuracy and precision of the fluorescence and temperature measurements determine the quality of HRM-PCR analysis, a thirdgeneration dye called EvaGreen was used.…”

Section: Discussionmentioning

confidence: 99%

“…Target sequences are first amplified using quantitative PCR (qPCR) in the presence of fluorescent dye, intercalating double-stranded DNA (dsDNA). The use of third-generation binding dyes (e.g., EvaGreen and SYTO9) ensures complete dsDNA saturation without inhibiting PCR [18,19]. Immediately following qPCR, the amplicon is gradually denatured by increasing the temperature in the same real-time instrument [19].…”

Section: Introductionmentioning

confidence: 99%

“…The use of third-generation binding dyes (e.g., EvaGreen and SYTO9) ensures complete dsDNA saturation without inhibiting PCR [18,19]. Immediately following qPCR, the amplicon is gradually denatured by increasing the temperature in the same real-time instrument [19]. As the temperature increases, the fluorescence emission values decrease since the denaturing amplicon releases the dye.…”

Section: Introductionmentioning

confidence: 99%

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Rapid Identification of Rhizobia Nodulating Soybean by a High-Resolution Melting Analysis

Jarzyniak,

Narożna

2024

Agronomy

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Soybean [Glycine max (L.) Merr.] is one of the most important and oldest crops. Due to its ability to form symbiotic interactions with nitrogen-fixing bacteria, it is a valuable source of nitrogen for agriculture and proteins for humans and livestock. In Europe, for instance, in Poland, the soybean cultivation area is still not large but is gradually increasing due to climate change. The lack of indigenous soybean microsymbionts in Polish soils forces the application of commercial strains to establish effective symbioses. Fast and reliable identification methods are needed to study the persistence, competitiveness, and dispersal of bradyrhizobia introduced as inocula. Our study aimed to apply real-time PCR coupled with high-resolution melting curve (HRM) analysis to detect and differentiate bacterial strains occupying soybean nodules. HRM-PCR was performed on crude extracts from nodules using primers specific for recA, a highly conserved nonsymbiotic gene. By comparing them with the reference strains, we were able to identify and assign Bradyrhiobium strains that had been introduced into field locations in Poland. In conclusion, HRM analysis was proven to be a fast and accurate method for identifying soybean microsymbionts and might be successfully used for identifying other legume-nodulating bacteria.

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“…However, it provides information on the methylation pattern, including the occurrence of mosaic [18] or monoallelic methylation [19]. In addition, it is highly sensitive and laborand cost-efficient [20]. A prerequisite for HRM is that the target region is amplified in the presence of a saturating DNA intercalating dye, e.g., EvaGreen.…”

Section: Introductionmentioning

confidence: 99%

Temperature-Wise Calibration Increases the Accuracy of DNA Methylation Levels Determined by High-Resolution Melting (HRM)

Zappe,

Cichna-Markl

2024

IJMS

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High-resolution melting (HRM) is a cost-efficient tool for targeted DNA methylation analysis. HRM yields the average methylation status across all CpGs in PCR products. Moreover, it provides information on the methylation pattern, e.g., the occurrence of monoallelic methylation. HRM assays have to be calibrated by analyzing DNA methylation standards of known methylation status and mixtures thereof. In general, DNA methylation levels determined by the classical calibration approach, including the whole temperature range in between normalization intervals, are in good agreement with the mean of the DNA methylation status of individual CpGs determined by pyrosequencing (PSQ), the gold standard of targeted DNA methylation analysis. However, the classical calibration approach leads to highly inaccurate results for samples with heterogeneous DNA methylation since they result in more complex melt curves, differing in their shape compared to those of DNA standards and mixtures thereof. Here, we present a novel calibration approach, i.e., temperature-wise calibration. By temperature-wise calibration, methylation profiles over temperature are obtained, which help in finding the optimal calibration range and thus increase the accuracy of HRM data, particularly for heterogeneous DNA methylation. For explaining the principle and demonstrating the potential of the novel calibration approach, we selected the promoter and two enhancers of MGMT, a gene encoding the repair protein MGMT.

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DNA melting analysis

Cited by 4 publications

References 90 publications

A simple PCR-based quick detection of the economically important oriental fruit fly, Bactrocera dorsalis (Hendel) from India

A simple PCR-based quick detection of the economically important oriental fruit fly, Bactrocera dorsalis (Hendel) from India

Rapid Identification of Rhizobia Nodulating Soybean by a High-Resolution Melting Analysis

Temperature-Wise Calibration Increases the Accuracy of DNA Methylation Levels Determined by High-Resolution Melting (HRM)

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