AccuMelt HRM SuperMix

AccuMelt HRM SuperMix maximizes differences in melt temperature and curve shape to allow discrimination of DNA sequence differences among different samples.

Features & Benefits

  • See subtle differences in sequence clearly – robust amplification ensures sufficient yield of products to generate discrete melt curves
  • Accurate genotype calling – comparable or better performance than TaqMan® Genotyping
  • Work with rare or precious samples – large range of template inputs possible
  • Specificity – works with lower Mg2+ concentration than other systems thus enhancing assay accuracy

 

AccuMelt HRM SuperMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Description

AccuMelt HRM SuperMix is a 2x concentrated, ready to use reaction cocktail for detection of genetic variations using high resolution melting (HRM) analysis. It includes all required components except for primers and DNA template. HRM is a closed tube, rapid and cost effective procedure for characterization of sequences differences immediately following PCR amplification. It is based on the melting (dissociation) behavior of a PCR product as it transitions from double stranded to single stranded DNA in the presence of a fluorescent dsDNA binding dye.

Details

  • Contents
    • 2 x reaction buffer containing optimized concentrations of MgCl2
    • dNTPs (including dUTP)
    • AccuStart Taq DNA Polymerase
    • SYTO® 9 green-fluorescent dye
    • stabilizers
    • Free Mg2+ = 0.8mM at 1x final concentration.
  • Storage & Handling
    Store components in a constant temperature freezer at -25°C to -15°C protected from light upon receipt. After thawing, mix thoroughly by gently vortexing tube contents. Centrifuge briefly to collect contents before using. For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.
  • Related Resources
    Product Manuals
    Product Flyers
    CofA (PSF)
    Publications
    The development and validation of a rapid genetic method for species identification and genotyping of medically important fungal pathogens using high-resolution melting curve analysis
    A.d. Alnuaimi, Molecular Oral Microbiology - 2014
    Abstract
    Accurate- rapid and economical fungal species identification has been a major aim in mycology. In this study- our goal was to examine the feasibility of a high-resolution melting curve analysis (HRMA) of internal transcribed regions ITS1 and ITS2 in ribosomal DNA (rDNA) for a rapid- simple and inexpensive differentiation of eight clinically relevant Candida species (Candida albicans- Candida glabrata- Candida parapsilosis- Candida krusei- Candida tropicalis- Candida guilliermondii- Candida dubliniensis and Candida lusitaniae). In addition- for the first time- we tested the applicability of HRMA to classify C. albicans strains into four previously described genotypes (A- B- C and D) using a primer set that spans the transposable intron region of 25S of rDNA. Type and unknown clinical oral isolates were used in this study and the melting curve analysis was compared with both amplicons' sequencing and agarose gel electrophoresis analysis. Real-time PCR and subsequent HRMA of the two described rDNA regions generated distinct melting curve profiles that were in accord with sequencing and gel electrophoresis analysis- highly reproducible- and characteristic of each of the eight Candida species and C. albicans genotypes. Moreover- results were obtained in 4 h and without the need for any post-amplification handling- so reducing time and cost. Owing to its simplicity and speed- this technique is a good fit for genotypic analysis of hundreds of clinical strains in large epidemiological settings.
    Development of a Screening System for the Detection of Chemically Induced DNA Methylation Alterations in a Zebrafish Liver Cell Line
    Eivind Farmen, Journal of Toxicology and Environmental Health- Part A - 2014
    Abstract
    Early molecular events with correlation to disease- such as aberrant DNA methylation- emphasize the importance of DNA methylation as a potential environmental biomarker. Currently- little is known regarding how various environmental contaminants and mixtures alter DNA methylation in aquatic organisms- and testing is both time- and labor-consuming. Therefore- the potential of an in vitro screening method was evaluated by exposing zebrafish liver cells (ZF-L) for 96 h to the nonmutagenic model substance 5′-azacytidine (AZA)- as well as a selection of environmental pollutants such as sodium arsenite (NAS)- 2-3-7-8-tetrachlorodibenzo-p-dioxin (TCDD)- 17alpha-ethinylestradiol (EE2)- and diethylstilbestrol (DES). Six single genes with reported and anticipated importance in cancer were selected for analysis. Methylation of gene promoter areas was monitored by bisulfite conversion and high-resolution melt (HRM) analysis after exposure to sublethal concentrations of the test compounds. Subsequently- results were validated with direct bisulfite sequencing. Exposure of ZF-L cells to 0.5 μM AZA for 96 h led to hypomethylation of genes with both low and high basal methylation indicating similarity to mechanism of action in mammals. Further- NAS- EE2- and DES were shown to induce significant alterations in methylation- whereas TCDD did not. It was concluded that cell line exposure in combination with HRM may provide an initial contaminant screening assay by quantifying DNA methylation alterations with high throughput capacity. In addition- the rapid determination of effects following contaminant exposure with this in vitro system points to the possibility for new in vivo applications to be useful for environmental monitoring.
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