High Resolution Melt (HRM) Assay

HRM is a simple and cost effective way to characterise the sequence of amplification products with no requirement for post-PCR processing. The basis of the HRM assays is the characteristic “melting behavior” of a particular DNA sample.

Real Time thermocyclers with the capacity for HRM analysis enable the precise characterisation of biological samples based on sequence length, GC content and/or DNA sequence heterogeneity. Therefore slight changes in sequence (for example class IV A/T SNPs) can be distinguished between samples (See Figure).

HRM is applicable, but not limited, to the following analyses:

• Detection of single base sequence variations (e.g. SNPs)
• Mutation discovery
• DNA mapping
• Identification of candidate predisposition genes
• Detect somatic mutations with a sensitivity of up to 3%
• Quantitate levels of transformed tissue.
• DNA methylation analysis
• Allelic prevalence
• HLA compatibility typing
• Species identification

HRM is made possible not only by specialised instrumentation and software, but also by the introduction of third generation fluorescent dsDNA dyes. Third generation dyes such as BEBO (TATAA Biocenter) have been successfully used for HRM analysis. These dyes exhibit low toxicity in an amplification reaction and can therefore be used at higher concentrations for greater saturation of the dsDNA sample. BEBO dye binds the minor groove of double-stranded DNA, and exhibits a strong fluorescence increase when it is in the dsDNA-bound form. It does not interfere with the PCR reaction.

SNP genotyping with Quantimix Easy HRM kit Melting profiles of homozygous G (green); homozygous T (blue); and heterozygous G/T (red).

Quantimix Easy HRM kit (with BEBO dsDNA binding dye)

Description:

QUANTIMIX EASY HRM KIT has been optimised to deliver maximum efficiency, precision, and sensitivity during nucleic acid amplification in real time using a third generation intercalating fluorescent dye. The master mix contains a thermo-stable polymerase (Biotools HotSplit DNA Polymerase) that minimises nonspecific product and/or primer-dimer formation, improving HRM sensitivity and specificity. In addition, the master mix which also includes the rest of components for real time amplification assay reduces set up time and liquid handling steps.

Contents:

• QUANTIHRM: It is a 2X ready-to-use solution that contains all necessary components for real time amplification assays: Biotools HotSplit DNA Polymerase, dNTPs, Reaction Buffer, and MgCl2 (final concentration: 4mM).

• 50 mM MgCl2 Solution: Used only for assays which require additional optimisation.

The intercalating fluorophore for HRM assay is not provided with the kit