Rotor-Gene Q MDx CE User Manual 02/2022
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High Resolution Melt Analysis
High resolution melt (HRM) analysis is an innovative technique that is based on analysis of DNA
melting. HRM characterizes DNA samples according to their dissociation behavior as they transition
from double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA) with increasing temperature
(see Figure below). A HRM instrument collects fluorescent signals with extremely high optical and
thermal precision, creating many application possibilities.
A typical HRM plot
. The melt curve plots the transition from the high fluorescence of the initial premelt phase, through the
decrease in fluorescence of the melt phase, to the basal level of fluorescence at the postmelt phase. Fluorescence decreases as
DNA intercalating dye is released from dsDNA as it melts into single strands. The midpoint of the melt phase, at which the
rate of change in fluorescence is greatest, defines the melting temperature (Tm) of the DNA under analysis.
Before performing HRM analysis, the target sequence must be amplified to a high copy number.
This is usually performed by PCR in the presence of a dsDNA intercalating fluorescent dye. The dye
does not interact with ssDNA but actively intercalates with dsDNA and fluoresces brightly when
intercalated. Change in fluorescence can be used to measure the increase in DNA concentration
during PCR and then to directly measure thermally-induced DNA melting by HRM. During HRM,
fluorescence is initially high because the sample starts as dsDNA. Fluorescence decreases as the
temperature is raised and DNA dissociates into single strands. The observed melting behavior is
characteristic of a particular DNA sample.
Using HRM, the Rotor-Gene Q MDx can characterize samples based on sequence length, GC
content, and DNA sequence complementarity. HRM can be used in genotyping applications, such
as analysis of insertions/deletions or single nucleotide polymorphisms (SNPs), or to screen for
unknown genetic mutations. It can also be used in epigenetics applications for detection and