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MeltDoctor High-Resolution Melting Reagents

Master mixes for screening variant sequences quickly and inexpensively

High Resolution Melting (HRM) analysis is a post-PCR technique used to identify genetic variation in nucleic acid sequences. HRM is often used to screen large numbers of potential variant sequences quickly and inexpensively as a precursor to other more laborious techniques such as sequencing. The resulting melt curve profiles can provide valuable information for genotyping studies in addition to many other applications, including mutation screening and methylation analysis.

 

Applied Biosystems MeltDoctor HRM reagents are specifically formulated to deliver sensitive and consistent results with HRM applications such as SNP analysis, methylation analysis, and mutation scanning.

HRM overview

HRM melt curve profile

In HRM, the region of interest is amplified in a reaction containing a double-strand DNA (dsDNA)–binding dye which fluoresces brightly only when bound to dsDNA. After PCR, reactions are slowly heated in a real-time instrument capable of capturing many fluorescent data points per change in temperature, with high precision. When the dsDNA dissociates (or melts) into single strands, the dye is released, causing a change in fluorescence.

The result is a melt curve profile characteristic of a particular sequence. The melt profile of a sample reflects the mix of amplicons present. Characteristics such as guanine-cytosine (GC) content, length, sequence, and heterozygosity will contribute to the melt curve of each amplicon.

HRM analysis differs from standard melt curve analysis (with SYBR Green dye, for example) in two ways:

  • Chemistry: HRM analysis uses a brighter dsDNA-binding dye that does not inhibit PCR at high concentrations.
  • Software: HRM analysis requires more specific software that uses specific data analysis algorithms and plots.

HRM applications

SNP genotyping

While TaqMan SNP Genotyping Assays are currently the gold standard for accurate and reproducible PCR-based genotyping, HRM is well suited for specific use cases. For example, when inherent sequence limitations prevent you from designing TaqMan probes, or when you are genotyping highly mutable samples where probe specificity may miss some species, HRM may be more successful. Similarly, consider HRM when you are analyzing many SNPs with low sample numbers, or analyzing nucleic acid variation in samples containing possibly unknown SNPs.

Other genotyping studies that can benefit from HRM include:

  • Genotyping in non-model organisms, where sequence knowledge is low
  • Association studies that require mapping of large regions
  • Checking the validity of National Center for Biotechnology Information (NCBI)-derived SNPs in a project population

Methylation analysis

HRM analysis can be used to detect methylation differences between genes; this is known as methylation-sensitive high-resolution melting (MS-HRM).

  • Quickly analyze the methylation status of specific genetic loci
  • MS-HRM followed by Sanger-based DNA sequencing can measure methylation levels as low as 0.1–2.0%

Genotyping & mutation scanning

HRM analysis can be used to scan for mutations in target genes to identify variant samples prior to sequencing analysis.
 

  • Easy, quick, cost-effective method for finding new genetic variations
  • Quickly and efficiently scan DNA samples from many individuals for minor genetic variations

MeltDoctor HRM Master Mix features

MeltDoctor HRM Master Mix contains all components needed for HRM-PCR (excluding template and primers). It is formulated for superior HRM performance across a wide range of genomic targets. Unlike some mixes on the market, MeltDoctor HRM Master Mix does not require additional mixing prior to use and was developed and optimized solely for HRM applications. Components of the master mix, provided in a convenient 2X mix, include:

Amplitaq Gold 360 DNA Polymerase

A highly purified DNA polymerase that provides hot-start performance, minimizing nonspecific product formation and enabling reactions to be set up at room temperature

MeltDoctor HRM Dye

Stabilized form of the fluorescent Invitrogen SYTO 9 double-stranded nucleic acid stain with optical and chemical properties important for high-performance HRM

dNTP blend including dUTP

Minimizes carryover contamination by allowing amplicon degradation by uracil-DNA glycosylase (UDG) in subsequent PCR reactions

Other high-quality components

Magnesium salts and other buffer components are precisely formulated to obtain optimal HRM results

Sample data

Mutation scanning using the HRM workflow.

Genomic DNA samples from three cell lines (HeLa, Raji, and Jurkat) were analyzed using HRM followed by DNA sequencing. Primers were designed to amplify 152 bp of exon 4 of the p53 tumor suppressor gene. Three genotypes are clearly distinguishable in the aligned HRM profile (left), and they were called accurately by the analysis software. Following HRM, the genotype of each sample (GC, GG, CC) was identified by sequencing (right).

MeltDoctor HRM solutions ordering information

MeltDoctor HRM Master Mix

MeltDoctor HRM Master Mix greatly simplifies your experimental setup as it contains everything you need. Just add your sample and PCR primer pair and you're ready to run your reaction.

 

MeltDoctor HRM Reagent Kit

The kit contains PCR components and MeltDoctor HRM dye required for high resolution melting analysis. Reagents are supplied unmixed in separate containers so that you can customize your experiments.

 

MeltDoctor HRM Positive Control Kit

This positive control kit contains components to demonstrate and troubleshoot HRM analysis, including forward and reverse PCR primers and three DNA templates representing alleles AA, AB and BB.

 

MeltDoctor HRM calibration reagents

Ready-to-use MeltDoctor HRM Calibration Plates contain all the components required for pure dye and HRM calibration, including MeltDoctor HRM Calibration Standard and MeltDoctor HRM Master Mix, reducing the complexity of getting started with HRM experimentation. They provide thermal and optical calibration required for accurate and reproducible discrimination between melt curves.

 

The MeltDoctor HRM Calibration Standard contains the DNA template and primers for researchers who prefer to prepare their own calibration plate for thermal and optical HRM calibration.

 

For Research Use Only. Not for use in diagnostic procedures.

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