Introduction to High Resolution Melt Analysis

Application Guide
High Resolution Melt Analysis

Contents Page

Introduction

Overview of the Melting Profile Principle

The Intercalating Dye

Non-saturating dyes
Saturating dyes
Release-on-demand dyes

4
4
4

Instruments and Software

Summary of the Workflow

Calibration of the instrument/Assay design/PCR/HRM/Data analysis

Analysis of Results
Amplification plots
Raw data melt curve
Normalization data
Difference graph

6
6
6
6

HRM Assay and Reagent Optimization

DNA quality and quantity
7
Primer design
8
Primer concentration and purification
8
Effect of MgCl2 on HRM analysis
9
Optimization of MgCl2 for KAPA HRM FAST
9
Amplicon length
9
Effect of PCR enhancers on HRM analysis
9
Common Problems and Assay Modification
Positioning of pre- and post-melt regions within the melt region
10
Non-specific amplification
11
Adjustment of annealing and extension time
12
Specific Applications
SNP genotyping
Amplification of two or more SNPs on a single amplicon
Mutation scanning and screening for heterozygosity

13
13
14

Typical Setup for Amplification and HRM with KAPA HRM FAST
Amplification and HRM using KAPA HRM FAST
15
Elimination of amplicon contamination prior to assay
15
Troubleshooting

16

Ordering Information

17

Version 1.10

Application Guide
High Resolution Melt Analysis

Introduction to High Resolution Melt Analysis

Introduction
3.2
3
2.8
2.6
2.4
3.2
2

-dF/dT

High Resolution Melting Analysis (HRM or HRMA) is a recently

developed technique for fast, high-throughput post-PCR analysis of
genetic mutations or variance in nucleic acid sequences. It enables
researchers to rapidly detect and categorize genetic mutations
(e.g. single nucleotide polymorphisms (SNPs)), identify new
genetic variants without sequencing (gene scanning) or determine
the genetic variation in a population (e.g. viral diversity) prior to
sequencing.
The first step of the HRM protocol is the amplification of the
region of interest, using standard PCR techniques, in the presence
of a specialized double-stranded DNA (dsDNA) binding dye. This
specialized dye is highly fluorescent when bound to dsDNA and
poorly fluorescent in the unbound state. This change allows the
user to monitor the DNA amplification during PCR (as in quantitative
PCR).
After completion of the PCR step, the amplified target is
gradually denatured by increasing the temperature in small
increments, in order to produce a characteristic melting profile; this
is termed melting analysis. The amplified target denatures gradually,
releasing the dye, which results in a drop in fluorescence. When
set up correctly, HRM is sensitive enough to allow the detection
of a single base change between otherwise identical nucleotide
sequences.
HRM uses low-cost dyes and requires less optimization than
similar systems based on TaqMan and fluorescence resonance
energy transfer (FRET) probes. Compared to these methods HRM
is a simpler and more cost-effective way to characterize multiple
samples.
This short guide describes the background theory of HRM,
in order to assist scientists in the development of HRM protocols.
Information is provided on how to optimize assays for maximum
sensitivity, how to analyse results obtained when using KAPA HRM
FAST, and how to troubleshoot.

1.8
1.6
1.4
1.2
1

0.8
0.6
0.4
0.2
0
71

72

73

74

75

76

77

78

79

80

81

82

Temperature C

83

84

85

86

87

88

89

90

Figure 1: Low Resolution Melt profile derivative plot (-dF/dT against T). The
steepest slope is easily visualized as a melt peak. In this example the Tm of the
amplicon is 77 C.

fluorescence data from low resolution melting curves can easily be

used to derive the Tm by plotting the derivative of fluorescence vs.
temperature (-dF/dT against T) as shown in Figure 1.
The principle of HRM is the same as a Low Resolution Melt,
except that the temperature difference between each fluorescence
reading is reduced. During a Low Resolution Melt curve analysis,
the temperature increases are typically in 0.5 C steps, but for HRM
this is reduced to 0.008 - 0.2 C increments. This allows a much
more detailed analysis of the melting behavior. HRM sensitivity and
reliability has been improved with the use of a variety of new dsDNA
Overview of the Melting Profile Principle
intercalating dyes; this is covered in more detail in the following
Amplification product melting analysis is not a novel concept. section. The introduction of HRM has revived the use of DNA
Post-PCR melt curve analysis using SYBR Green I dsDNA melting for a wide range of applications, including:
intercalating dye to detect primer-dimers or other non-specific
SNP genotyping
products has been performed for over a decade. Today this
Mutation discovery (gene scanning)
procedure is called Low Resolution Melting.
Heterozygosity screening
A Low Resolution Melt curve is produced by increasing the
DNA fingerprinting
temperature, typically in 0.5 C increments, thereby gradually
Haplotype blocks characterization
denaturing an amplified DNA target. Since SYBR Green I is only
DNA methylation analysis
fluorescent when bound to dsDNA, fluorescence decreases as
DNA mapping
duplex DNA is denatured. The melting profile depends on the length,
Species identification
GC content, sequence and heterozygosity of the amplified target.
Viral/bacterial population diversity investigation
The highest rate of fluorescence decrease is generally at the
HLA compatibility typing
melting temperature of the DNA sample (Tm). The Tm is defined
Association study (case/control)
as the temperature at which 50% of the DNA sample is doublestranded and 50% is single-stranded. The Tm is typically higher for
DNA fragments that are longer and/or have a high GC content. The
3

Application Guide
High Resolution Melt Analysis

The Intercalating Dye

There are various types of dsDNA intercalating dyes, which
have distinctly different properties. The requirements of the dyes
used for HRM are different from dyes typically used for standard
quantitative PCR (qPCR) assays. Factors critical in qPCR, such
as the signal-to-noise ratio and amplification efficiency, are not
essential requirements for HRM. Instead, the dye must provide
detailed information on the melting behavior of an amplified target.
Ideally the dye should not bind preferentially to pyrimidines or
purines, change the Tm of the amplicon, or inhibit DNA amplification.
This section briefly describes the three main classes of dsDNA
binding dyes that are currently available.
Non-saturating dyes. SYBR Green I is the most common
non-saturating dsDNA intercalating dye. It is generally unsuitable
for most HRM applications. At high concentrations, SYBR Green I
stabilizes the duplex DNA and inhibits the DNA polymerase. To allow
reliable amplification, low concentrations of SYBR Green I must
therefore be used. At lower concentrations, SYBR Green I is able
to redistribute from the melted regions of single-stranded DNA back
to the regions of dsDNA, which results in poor base-difference
discrimination, as detailed in Figure 2. To overcome this limitation,
saturating dyes have been developed.

Saturating dyes. A new class of dsDNA intercalating dyes

that do not inhibit DNA polymerases, or alter the Tm of the product,
have recently been developed; these dyes can be added at higher
concentrations than non-saturating dyes, ensuring more complete
intercalation of the amplicon. The dye is not able to redistribute
during melting because the dsDNA is saturated. More precise
examination of the melting behavior is therefore possible, as
indicated in Figure 2. Dyes such as SYTO9 and LCGreen can be
used at the saturating concentrations required for HRM.
Release-on-demand dyes. The release-on-demand class
of dyes, which include EvaGreen, can be added at non-saturating
concentrations. This is due to the novel method of fluorescence
emission, where the fluorescent signal is quenched when the dye is
free in solution. Upon binding to duplex DNA, the quenching factor
is released and the dye emits high fluorescent signal. This allows
non-saturating concentrations of the dye to be used, ensuring that
there is no PCR inhibition, whilst the unique dye chemistry provides
highly sensitive HRM analysis.

Non-saturating dye

Saturating dye

Release on demand

eg. SYBR

eg. SYT09

eg. EvaGreen

Melting

No fluorescence change

Melting

Drop in fluorescence

Drop in fluorescence

Figure 2: Non-saturating, saturating and release-on-demand dsDNA intercalating dyes. Melting of the duplex as the temperature increases releases the intercalated
dyes. At non-saturating concentrations the dye rapidly rebinds to regions that remain double stranded; consequently there is no drop in fluorescence. Saturating and releaseon-demand dyes do not redistribute from the melted regions of single-stranded DNA back to dsDNA, resulting in a reduction of fluorescence. This difference gives dyes such
as EvaGreen the high sensitivity required for HRM analysis.

Application Guide
High Resolution Melt Analysis

Instruments and software

Summary of the workflow

The power of any HRM analysis depends Pre-assay preparations

upon the sensitivity of the instrument and
the nature of the PCR reagents used. Certain Step 1 Calibration of the instrument
instruments have been designed specifically
The correct calibration of the instrument prior to every use is
for HRM analysis. These can be summarized
essential, because different dyes have different emission spectra.
into two distinct classes:
It is recommended that different manufacturers' products are not
used in the same run. For each set of reagents, the calibration of the
1. Block based instruments Samples
instrument may require adjustment. The manufacturers handbooks
are placed in a block for cycling and a
describe how to change the filter set to the correct settings prior to
scanning head or stationary camera is
PCR and HRM.
used for detection.
Step 2 Assay design
2. Rotary Samples reside in a single
Proper assay design is critical for successful HRM analysis. The
chamber and spin past an optical
quality of DNA, correct choice of primers, choice of reagents and
detector.
optimization of the protocol all play key roles in obtaining the best
results possible. The section HRM Assay and Reagent Optimization
(Page 7) provides detailed instructions on how to design an
There
are
advantages
and
assay. KAPA HRM FAST PCR Kits are designed for reliable, fast
disadvantages with each class, and the user
amplification and maximum sensitivity and contain a ready-to-use
should choose the type of instrument that
Master Mix that simplifies the design stage.
best suits their needs. As a general rule, for
high sensitivity and reproducibility a rotarybased instrument should be used, whilst Running the assay (Steps 3, 4 and 5 should be completed on the same instrument if
for high-throughput and ease-of-handling, possible.)
block-type instruments are optimal. The
HRM run can vary considerably between Step 3 PCR
instruments; some take considerably longer
It is recommended that real-time PCR is conducted; amplification can
than others. Every instrument should be
then be monitored and any troubleshooting that may subsequently
calibrated regularly (as recommended by
be required is made easier. If an assay has already been optimized,
the manufacturer, typically every 6 months),
the PCR may be performed on any standard thermocycler (ramp
kept in a clean area on a secure and stable
speeds may vary between instruments and the annealing/extension
benchtop, and checked daily for dust buildtimes may have to be adjusted) and the samples then transferred to
up around the optics.
a real-time thermocycler capable of HRM.
The computer hardware and software
must be capable of handling the large Step 4 HRM
quantities of data usually generated during
The HRM step is performed after the final cycle of the PCR. Usually,
a HRM experiment. Analysis of the data is
various settings can be adjusted; these include the start and end
relatively easy with the appropriate software.
temperatures, and the temperature increments and hold times
HRM specific software uses various
between data acquisitions, typically 0.008 0.2 C steps, with a 2
algorithms to analyze the data and display the
second hold-time. Some instruments, however, do not allow such a
results in easily accessible formats to help
high degree of flexibility and have a single protocol that cannot be
discriminate between sequence variants.
adjusted. The section Typical setup for amplification and HRM with
Some of these algorithms are described in
KAPA HRM FAST (Page 15) describes how to set up a PCR and
the following sections. Additional software
HRM protocol.
packages, designed for more detailed
analysis, are available from the instrument Post-assay analysis
manufacturer.
Step 5 Data Analysis
The HRM data is analyzed by software specifically designed for the
purpose (usually supplied by the thermocycler manufacturer). The
section Analysis of Results (Page 6) details how to select pre- and
post-melt regions for creating normalized and difference plots. The
most common problems encountered and how to solve them are
summarized in Troubleshooting (Page 16).

Application Guide
High Resolution Melt Analysis

Analysis of results

Raw data melt curve. The data collected during the HRM
analysis has a range of initial (pre-melt) fluorescence readings. This
variance makes it difficult to properly analyze results, even though
different genotype groups may be visible. Figure 3A illustrates how
the selection of pre-and post-melt regions is used to align data.
Pre- and post-melt regions must be selected for each primer set,
by positioning the parallel double-bars as shown. It is important to
adjust these bars so that the melt region is not selected. If the preor post-melt regions are not clearly defined, it is possible to repeat
the HRM run only (without repeating the amplification step), and
adjust the temperature range as required.

Normalization region 2

90

Fluorescence variance

80

Fluoresence

70
60
50
40
30
20
10
0

70

71

72

73

74

75

76

74

74.5

75

75.5

76

76.5

74

74.5

75

75.5

76

76.5

77

80

82

78

79

77

77.5

78

78.5

79

79.5

80

77

77.5

78

78.5

79

79.5

80

Temperature (C)

81

83

84

100
90
80
70
60
50
40
30
20
10

Temperature (C)

8
6
4
2

Nomalized minus (A/A)

Difference graph. Some HRM software applications allow

calculation of the difference plot. This is achieved by subtracting
the normalized fluorescence data of a user-defined genotype from
that of each of the other samples in the HRM analysis. In Figure 3C,
the A/A genotype has been selected as a baseline (any genotype
can be selected, but usually one of the homozygotes is used). The
position of each sample relative to the baseline is plotted against the
temperature. This form of HRM analysis is an aid for visualizing the
Normalization Data.
Automatic calling of genotypes of sequence variants can usually
be performed with the software; sometimes confidence ratings are
given. However, checking the difference to confirm the genotype is
recommended.

Post-melt

100

Normalization data. If the data is normalized correctly, it

will appear as shown in Figure 3B. This is termed Normalization
Data. Here the fluorescence variance seen in Figure 3A has been
eliminated, and only the temperature range between the outer bars
of the pre- and post-melt regions is shown. The genotypes are now
more distinct, but the two homozygote samples (in red and blue) are
still difficult to distinguish.

Melt

Normalization region 1

Amplification plots. Reproducibility of amplification is more

important for HRM analysis than the efficiency of amplification
(as compared to qPCR). For example, HRM analysis can still
be performed if the product amplifies later than expected
(i.e. due to limitations in primer design), provided that all the samples
are consistent, the no template control (NTC) does not amplify and
the product is specific.

Pre-melt

Nomalized Fluoresence

With the correct software, data analysis is typically

straightforward, allowing multiple samples to be analyzed
simultaneously. It is important to know what to look for when
interrogating results; unforeseen errors made during setup can be
identified at this point. Low resolution melt plots are often viewed
as a derivative plot (dF/dT against T); however, HRM raw melting
curves must be analyzed differently. This section concerns the
analysis of HRM results; a 124 bp product covering a Type IV SNP
(rs641805) is used as an example. The most common problems
encountered and their solutions are also discussed.

0
-2
-4
-6
-8

-10
-12
-14

-16

Temperature (C)

Figure 3: Analysis of HRM data from a type 4 SNP (A/T). Different genotypes are
highlighted in different colors. A/A = red, A/T = green, and T/T = blue. A. Raw data
showing the pre-melt, melt and post-melt regions. Notice the fluorescence variance
and the positioning of the pre- and post-melt identification bars. B. Normalization Data
derived from the raw data plots in A. Positioning of the pre-melt, and post-melt bars
provides a more detailed view of the melt region. C. Difference Graph derived from the
Normalization Data.
6

Application Guide
High Resolution Melt Analysis

HRM assay and reagent optimization

Due to the highly sensitive nature of HRM analysis, proper
assay design is critical. Particular attention should be given
to primer design, PCR reagents and cycling conditions. Small
differences in the initial setup can greatly affect the final results.
Factors such as genomic DNA (gDNA) quality, primer design, MgCl2
concentration and carryover of inhibitors will all affect the melting
behavior. Achieving specific amplification is critical to the success
of an assay, since any non-specific amplification will greatly impair
the melt analysis. The following sections address each of these
concerns.
DNA quality and quantity. One of the major factors that can
detract from the quality of results is the carryover of buffer from
template DNA samples/preparations. Unsuitable DNA purification
techniques can exacerbate this problem. Salt carryover can subtly
change the melting behavior of the PCR product, and can result
in low sensitivity, poor reproducibility and incorrect genotype calls.
Buffer carryover from the template DNA will not only modify the Tm
and melting behavior during the HRM, but can also encourage nonspecific amplification during the PCR.
For optimum results the following is recommended:
Ideally, all DNA samples to be analyzed (including genotype
controls) should be extracted using the same DNA extraction
method or kit.
Some kits use high concentrations of salt for elution of DNA
from columns. All template samples should be eluted with low
salt buffers, ideally 10 mM Tris-HCl, pH 8.5.
The DNA sample should be as concentrated as possible after
purification. The template can then be diluted with 10 mM TrisHCl, pH 8.5 prior to use. This helps to reduce buffer variation
between samples.
Addition of a low volume of template DNA will reduce the
carryover salt in the reaction; ideally the lowest volume of
template that can be accurately added should be used (1 - 2 l).
All samples should have a similar final concentration of DNA in
each assay. Table 1 details the recommended concentration
of DNA for different types of template. At low template
concentrations, there is a greater chance of incorporating a
mutation early in the PCR which will affect the melting behavior;
high concentrations of DNA will result in high background
fluorescence due to increased intercalation of dye into dsDNA.
Positive control(s) for all genotypes should be included
where possible, preferably at the same concentration as their
corresponding test samples. Control DNA should also be eluted
and/or diluted in the same buffer as the samples.

Table 1
Type of DNA

Genomic DNA (gDNA)

Recommended amount
for HRM analysis/reaction
10 ng - 100 pg

Plasmid DNA (1 10 kb)

1 ng - 10 fg

Amplicon DNA

10 pg - 1 fg

Related product: KAPA Express Extract

Extraction of HRM-ready DNA by a quick and simple
protocol
KAPA Express Extract is a novel thermostable protease and
buffer system formulated for the extraction of PCR-ready DNA
in 10 minutes, from a broad range of tissue types. Combining
KAPA Express Extract and KAPA HRM FAST allows for the
extraction, amplification and HRM in less than 2 hours.
In order to minimze the effect of salt carryover, the use of
KAPA Express Extract Buffer at a final concentration of 0.5X is
recommended. For optimum performance, 1 l of the DNA extract
is added directly to each KAPA HRM FAST reaction.
DNA extracted from samples such as buccal swabs and hair
follicles (which contain a lower concentration of inhibitors) may
be used directly, with no additional purification required. For more
complex samples, such as formalin-fixed, paraffin-embedded
(FFPE) tissues, the carryover of contaminants can affect the
background fluorescence, and will vary between samples. In these
cases the extracted sample should be diluted 1:10 1:100 with
10 mM Tris-HCl pH 8.5 prior to amplification/HRM.
For more details see http://www.kapabiosystems.com

Application Guide
High Resolution Melt Analysis

HRM assay and reagent optimization (cont.)

Primer design. The design of primers for HRM analysis can be
a challenge, due to the fact that small amplicon sizes are required for
maximum sensitivity. The short amplicon lengths required for HRM
restricts the number of potential primer locations; well-designed
primer sets will improve the quality of the final result. It is common
practice to use a primer design software program such as Primer3
or Primer3Plus. This software assists in positioning primers such
that specific regions are included or excluded, and amplicon size
can be specified. Potential primer sets and full-length amplicons
can then be tested for specificity using a BLAST search. Factors
such as the position of the primer, the need for a GC region on the
3 end, the presence of any secondary structures in the primers
or target, and the predicted specificity of amplification should be
carefully considered. Figure 4 shows an example of the input page
of the Primer 3 design package, when used to design primers for
amplification of a human SNP.

amplification product. This should be examined using

appropriate software. Amplification of any secondary structure
within the product may result in unusual melting profiles.
Primers may need to be redesigned to avoid areas of secondary
structure.
Amplicon size. This must be optimal for the specific application,
typically between 50 - 300 bp.

Primer concentration and purification. The primer

concentration should not greatly affect the overall HRM result.
Minor changes are possible, but are likely to be amplicon specific.
Primers that have been purified using standard desalting methods
can be used for many HRM applications. The primary consideration
is the carryover of salt, and the effect this has on HRM analysis. To
minimize the impact of batch to batch variability often observed in
standard desalted primers, low primer concentrations (0.05 or 0.1 M
final) can be used. Despite the expense, HPLC-purified primers
After the primers have been designed, the following factors must are always recommended to ensure maximum reproducibility and
sensitivity when attempting to differentiate products with very
be checked:
similar melting profiles, e.g. Type IV SNPs
The presence of a GC clamp on the 3 end. This is not essential,
At high primer concentrations, the chance of non-specific
but usually improves amplification.
The specificity of the primers and the presence of known amplification is increased due to mispriming. The recommended
sequence variations. A BLAST search with both primer sets primer concentration when using KAPA HRM FAST is between 0.05
and the expected amplification product should be performed. and 0.5 M. An example of non-specific amplification is provided in
section Non-specific amplification on Page 11.
If possible, sequence variations should also be checked.
The secondary structure of the primers and the expected

Sequence input

Target SNP
(with square
brackets)

Select
appropriate library
type to reduce
mispriming

Product
size (bp)

Figure 4: Design of primers for amplification of a human SNP using Primer 3 (http://frodo.wi.mit.edu/primer3/). The target SNP is shown in square
brackets (e.g. ... TTAA[A]TGTC...) and the product size is set to 50 300 (bp). Selection of the mispriming library HUMAN helps reduce the chances of non-specific
amplification.
8

Application Guide
High Resolution Melt Analysis

HRM assay and reagent optimization (cont.)

Effect of MgCl2 on HRM analysis. The concentration of MgCl2
in PCR is known to greatly affect amplification efficiency. For some
targets, adjusting the MgCl2 concentration will result in reduced
non-specific amplification, allowing clearer distinction of sequence
variations; however, MgCl2 concentration has other, notable
effects on HRM. It is important to realize that the optimum MgCl2
concentration for amplification efficiency is not necessarily optimal
for HRM sensitivity. The effect of MgCl2 is shown in Figure 5. As
the MgCl2 concentration is increased, the HRM difference graphs
change distinctively.

Nomalized minus T/T

the more difficult it is to obtain clear discrimination between

sequence variants. For single base changes or SNPs, and for single
base insertions/deletions, it is recommended that amplicons be 50
300 bp. The clearest discrimination is usually seen with the shortest
amplicons. As the amplicon size increases, a more complex
melting pattern can develop, making it more difficult to distinguish
between single nucleotide variants. The use of smaller amplicons
may result in low fluorescence values, presumably due to reduced
dye incorporation. However, this is usually not a problem unless the
assay is run alongside a longer amplicon assay.
When screening for unknown sequence differences, longer
Optimization of MgCl2 for KAPA HRM FAST. KAPA HRM amplicons (typically 200 500 bp) can be used. This is useful in
FAST Master Mix does not contain MgCl2, and it is recommended gene scanning or determining the variation within a population (e.g.
that the MgCl2 concentration be optimized for each new assay viral) and reduces the number of primer sets required.
prior to large scale analysis. This will help minimize non-specific
Effect of PCR enhancers on HRM analysis. PCR enhancers
amplification, assist in separating genotypes and result in a greater
likelihood of differentiating sequence variations. An initial titration of are commonly added to end-point PCR in order to help reduce
MgCl2 at 1.5, 2.5 and 3.5 mM, using the 25 mM MgCl2 provided in non-specific amplification, reduce the number of required cycles
the kit, is recommended and further optimization may be required. If and increase yield. These enhancers function by assisting the
this is not possible, a final concentration of 2.5 mM is recommended melting and annealing of primers and templates, and can affect
HRM performance. For GC-rich amplicons, the addition of DMSO
for all assays.
appears not to affect the HRM significantly, and is recommended if
Amplicon length. The optimal length of an amplicon is amplification is difficult. Overall, the addition of PCR enhancers is
dependent upon the nature of the assay. The longer the amplicon, not recommended as they usually detract from HRM performance.

23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
-1
-2
-3
-4
-5
-6
-7
0

Increasing MgCl2 concentration

70

70.5

71

71.5

72

72.5

73

73.5

74

74.5
75
75.5
Temperature C

76

76.5

77

77.5

78

78.5

79

79.5

80

Figure 5: Effect of increasing MgCl2 on melting behavior. Three Difference Graphs are overlayed to demonstrate the dependence of amplicon melting on MgCl2 concentration.
The effect of MgCl2 is most pronounced in the heterozygote samples (blue), a result of the magnesium stabilizing the annealing of mispairs. The MgCl2 concentrations are
1.5 mM, 2.5 mM and 3.5 mM final. G/G = green, G/A = blue, and A/A = red.
9

Application Guide
High Resolution Melt Analysis

Common problems and assay modification

Pre-melt

Melt

100
90

Fluorescence variance

80

60
50
40

Normalization region 1

Fluoresence

70

30
20
10
0

70

71

72

73

74

75

76

77

78

79

Temperature (C)

80

81

82

83

84

100

Nomalized Fluoresence

90
80
70
60
50
40
30
20
10
0

74

74.5

75

75.5

74

74.5

75

75.5

76

76.5

77

77.5

78

76

76.5

77

77.5

78

Temperature (C)

4
2
0

Nomalized minus (A/A)

Positioning of pre- and post-melt regions within the melt

region. When the pre- and/or post-melt regions are positioned
within the melting region of the melting curve raw data (as shown
in Figure 6A), the subsequent Normalization Data and Difference
Graphs obtained are of poorer quality (as shown in Figure 6B and
6C). Placing the post-melting parallel bars within the melting region
causes the difference between the two homozygotes to become
smaller and less distinct. The problem is solved by repositioning
the pre- and post-melt normalization regions outside of the melting
region. If the temperature range of the HRM protocol starts or ends
within the melting range, repeat the HRM protocol (without repeating
the amplification step) using an appropriately wider range to ensure
the pre-melt or post-melt regions are clearly visible.

Post-melt

Normalization region 2

When analyzing results, it is important to know what to expect

when something goes wrong. Identification of a problem is vital
to help the user to re-design the protocol or alter the parameters
of analysis to obtain maximum sensitivity and reliability. In this
section, a series of experimental results that have been analyzed
inaccurately, or are from poorly optimized protocols, is shown.

-2
-4
-6
-8

-10
-12

-14
-16

Temperature (C)

Figure 6: Positioning the post-melt region within the melt curve and subsequent
analysis. A. Melting curve raw data showing the position of the post-melt identification
bars within the melt region. B. The subsequent Normalized Data when the post-melt
identification bars are located within the melt region. C. The Difference Graph does
not show as clear differentiation when compared to Figure 3C. Genotypes are typically
still distinguishable, but with a large number of samples, the likelihood that some will
be mis-called is greatly increased. Similar results are obtained when the temperature
range of the HRM protocol ends or begins within the amplicon melting range.
10

Application Guide
High Resolution Melt Analysis

Common problems and assay modification (cont.)

Annealing/extension at 58C
18
16

100
Normalized minus T/T

14

90

Normalized Fluoresence

Non-specific amplification. Non-specific amplification

products generated during PCR may lead to modified melting
behavior of the target amplicon. This greatly affects the quality of
the final results. It is therefore important to recognize the features
of a HRM plot that are indicative of non-specific amplification. To
demonstrate the effect of non-specific products, Figure 7 shows
the Normalization Data and Difference Graphs from a HRM assay
of a Type I SNP, using two different annealing temperatures.
Although the three genotypes can still be distinguished at the lower
annealing/extension temperature, non-specific products reduce
the difference between the variants, increasing the possibility of
mis-calling among samples. It is important to note that in some
cases, non-specific amplification may occur as a consequence of
a single nucleotide difference, which would restrict non-specific
amplification to templates of a single genotype.

80
70

12
10
8
6
4
2
0
-2

60

-4
77

78

79

80

50

81

82

83

Temperature (C)

84

85

86

40
30
20
10
77

78

79

Various techniques can be employed to reduce non-specific

amplification, including the following:

80

81

82

83

Temperature (C)

84

85

86

Annealing/extension at 53C
18

Normalized minus T/T

14

90

Normalized Fluoresence

Increasing the annealing/extension temperature.

Reducing the primer concentration.
Optimizing the MgCl2 concentration.
Using a 3-step protocol with a reduced annealing time and
additional extension step at 72 C (for longer amplicons typically
used in gene scanning or diversity studies (e.g. 200 500 bp)).
Re-designing of the primers.

16

100

80
70

12
10
8
6
4
2
0
-2

60

-4
77

78

79

80

50

81

82

83

Temperature (C)

84

85

86

40
30
20
10
77

78

79

80

81

82

Temperature (C)

58C
bp

c/c

c/t

83

84

85

86

53C
t/t

ntc

c/c

c/t

t/t

ntc

300
200
150

100

Figure 7: Effect of non-specific amplification on HRM analysis. HRM of a 106 bp

product containing Type I SNP rs2238289 (C:T); this SNP is part of the haplotype
involved in determining eye color. The target was amplified using a 30 sec annealing/
extension at either 58 C or 53 C. C/C = red , C/T = green and T/T = blue. A. 58 C
Normalization Data and Difference Graphs (inset). B. 53 C Normalization Data and
Difference Graphs (inset). C. Samples from each annealing/extension temperature
and genotype were run on a 4% agarose gel to determine if non-specific products
were produced. NTC = No Template Control, M = KAPA Universal Ladder. An
additional very faint band at ~200 bp was present when the annealing/extension
temperature was at 53 C.
11

Application Guide
High Resolution Melt Analysis

Common problems and assay modification (cont.)

Adjustment of the annealing/extension time. Obtaining full length products during amplification helps ensure consistent melting
behavior in the analyzed samples. When the annealing/extension time is too short, incomplete amplicons (formed when the DNA
polymerase does not reach the end of the target) can have a significant effect on the final HRM analysis. To demonstrate the effect of the
annealing/extension time on the Normalization Data and Difference Graphs, Figure 8 shows the analysis of a 183 bp product covering
a Type I SNP, using two different annealing/extension times. The derivative plots are indistinguishable; however, the Normalization Data
and Difference Graphs are distinctly different, with the longer annealing/extension resulting in better separation of the three genotypes.
There are various factors that will affect the optimal annealing/extension time for HRM analysis. These include the length, sequence
and secondary structure of the target. Longer annealing/extension times ensure that full length amplicons are produced, but can also
increase the possibility of mispriming and non-specific amplification.
20 sec annealing/extension

40 sec annealing/extension
4.5

4.5
4

100

100

3.5

3.5
3

dF/dT

dF/dT

3
2.5

90

2.5

90

80

80

0.5

0.5
0

0
72

73

74

75

76

77

Temperature (C)

78

79

80

81

71

82

Normalized Fluoresence

Normalized Fluoresence

71

70
60
50
40

10

10
76

76.5

77

Temperature (C)

77.5

78

78.5

79

30

76

77

Temperature (C)

78

79

80

81

82

74

74.5

75

75.5

76

76.5

77

77.5

78

78.5

79

74

74.5

75

75.5

76

76.5

77

77.5

78

78.5

79

Temperature (C)

30

25

25

20

20

Nomalized minus (T/T)

Nomalized minus (T/T)

75

40

20

75.5

74

50

20

75

73

60

30

74.5

72

70

30

74

1.5

1.5

15

10

15

10

0
0
-5
-5
-10
-10
74

74.5

75

75.5

76

76.5

77

Temperature (C)

77.5

78

78.5

79

Temperature (C)

Figure 8: The effect of annealing/extension time on HRM analysis. A. Normalization Data and Difference Graph (inset, C/C = red, C/T = blue and T/T = purple) of
Type I SNP rs11755 (C/T), with a 20 sec annealing/extension time. B. Difference Graph of the Normalization Data in A. C. Normalization Data and Difference
Graph (inset, C/C = red, C/T = blue and T/T = purple) of Type 1 SNP rs11755 (C/T), with a 40 sec annealing/extension time. D. Difference Graph of the Normalization Data in C.
The data obtained shows more consistent melting profiles between samples when the annealing/extension time is increased.
12

Application Guide
High Resolution Melt Analysis

Specific applications
Table 2

The generation and comparison of amplicon melting profiles is

a powerful tool in molecular biology. HRM is especially useful since
it requires no additional setup (aside from PCR), and melt profiles
are obtained directly after amplification, supplying information not
obtained from low resolution melt profiles. It is a closed tube assay
that allows genotyping without the risk of cross-contamination.
Additionally, the HRM step can be repeated several times if required
(although the intercalating dye will gradually degrade) to verify results
or to test modified settings. After the HRM is complete, the samples
can still undergo sequencing (after cleanup) or can be run on an
agarose gel. In this section, the most common applications of HRM
are described, with focus on assay development and optimization.

Personalized medicine
Disease susceptibility
Drug sensitivity or resistance
DNA variants of complex haplotypes
Endangered species population genetics
Crop and livestock breeding

Base
Change

Typical change
in Tm

Occurrence
in human
genome

C/T and G/A

Large (<0.5 C)

64%

II

C/A and G/T

Large (<0.5 C)

20%

III

C/G

Small (0.2 - 0.5 C)

9%

IV

A/T

Very Small (<0.2 C)

7%

105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5

Normalized Fluoresence

SNP Genotyping. Single nucleotide polymorphism (SNP) is

the term used to describe a DNA sequence variation of a single
nucleotide between members of a species or paired chromosomes
in an individual. These variations in the DNA sequence can affect
how individuals in a population develop diseases and respond to
pathogens, chemicals, drugs, vaccines, and other agents. Most
SNPs operate in conjunction with multiple other SNPs to manifest
an effect; this means that large populations of individuals need to be
examined if detailed and meaningful results are required. In humans,
SNPs make up about 90% of all genetic variation and occur (on
average) every 100 to 300 bases along the 3 billion base genome.
SNP studies can play a role in a variety of fields of study, including:

SNP
Class

72

73

74

75

76

77

78

79

80

81

82

72

73

74

75

76

77

78

79

80

81

82

Temperature (C)

24

Amplification of two or more SNPs on a single amplicon. In

some cases, two or more SNPs can be located very close together
making it impossible to design a primer to amplify a single SNP. It is
however possible to amplify a region containing multiple SNPs and
perform HRM analyses. This will result in several melting profiles,
yet the different genotypes of both SNPs can be determined if all the
necessary genotype controls are included. The addition of a probe
may help distinguish the individual SNPs if necessary.

20
18
16

Nomalized minus (A/A)

Usually, a short segment around the SNP in question is

amplified; the amplicon is typically 50 300 bp in length. The longer
the amplicon, the more difficult it is to distinguish the different
genotypes, and the greater the chance of including other, unwanted
SNPs that will affect discrimination. Ideally, the SNP is located close
to the middle of the amplicon. An example of HRM analysis of a
human SNP is shown in Figure 9. The expected shift in Tm for each
type of SNP (or any sequence variation) is detailed in Table 2.

22

14
12
10
8
6
4
2
0
-2
-4

Temperature (C)

Figure 9. HRM Analysis of the rs12913832 SNP (G/A) near the OCA2 gene that
is functionally linked to blue or brown eye color. A 124 bp product was amplified
from 1 ng of pure genomic DNA using KAPA HRM FAST with primers designed
using Primer3. The three genotypes are clearly distinguished in this assay. The G/G
homozygote melts at the highest temperature (red) whilst the A/A homozygote melts
at approx 1 C lower (green). The heterozygote is usually easiest to distinguish with an
altered melt profile (blue).
13

Application Guide
High Resolution Melt Analysis

Specific applications (cont.)

If there are known hot spots or sites of interest, shorter
Mutation scanning and screening for heterozygosity. The
products (100 200 bp) are preferable over these regions.
identification of sequence variants without prior knowledge of
the identity or location of the variation usually involves screening
Figure 10 shows an example of the location of nine different
of entire genes and their upstream/downstream transcriptionregulating regions. To efficiently screen the whole target area, assay amplicons covering a 3 kb gene, including exons, introns and
design is very important. In general, the following guidelines should neighboring elements. The average amplicon size is 333 bp,
but shorter products are used in a region where there is a high
be followed:
concentration of variations.
Amplicons are typically longer than those used for SNP
It is possible to detect sequence variations with longer products
detection, ranging from ~250 bp to 500 bp in length. A longer
amplicon length ensures complete coverage of the target (e.g. >500 bp); however, the variation in the melting behavior of
region with a reasonable number of primer sets. The length of the product decreases as amplicon length increases. Longer
the amplicon is selected depending on which mutation(s) are to amplicons are more likely to form complex secondary structures,
be detected (e.g. a Type I SNP is easier to detect than a Type IV which may result in multiple melt peaks. Such multiple peaks are
also indicative of non-specific amplification occurring during the
SNP, and so a longer product can be amplified).
Primers should not cover regions with known sequence PCR, so products may need to be analyzed by gel electrophoresis
variations; this can lead to preferential amplification of one to confirm specificity.
sequence variant over another.
Amplicons should overlap by at least 25-30 bp (to cover the
priming regions and ensure that all parts of the target region
are amplified).

600 bp

200 bp

150 bp

200 bp

400 bp

Figure 10: Design of amplicons for scanning a 3 kbp gene and surrounding regions for sequence variations. A gene consisting of 5 exons (blue) requires several
different primer sets to ensure complete coverage of the target region. Amplicons (orange) are designed to overlap, with shorter products targeting regions of interest, or known
mutation hot-spots (mutations shown by red triangles).

Extra information:
Heterozygote samples

3.6
3.4
3.2
3
2.8
2.6
2.4

dF/dT

With heterozygote samples, a composite of heterozygote

(resulting from mispairing) and homozygote duplexes are created.
This results in a mixture of four different melting profiles, all
combined into one fluorescent signal output. The melting profile plot
of a heterozygote sample (in green) often displays a double peak
compared to the single peak of the homozygotes (red and blue).
The relatively unstable mispaired heteroduplexes melt at a lower
temperature than the correctly paired samples, so heterozygotes
are easily identified. Addition of higher concentrations of MgCl2 can
help increase the number of mis-paired heterozygote samples. This
is especially useful when performing gene scanning applications.

2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

71

72

73

74

75

76

77

78

79

80

81

82

83

84

14

Application Guide
High Resolution Melt Analysis

Typical setup for amplification and HRM with KAPA

HRM FAST PCR Kits
Table 3

KAPA HRM FAST PCR Kits contain the release-on-demand

green fluorescent dye EvaGreen (Biotium), which was selected
to allow maximum sensitivity with minimal inhibition. The buffer
is specifically formulated to maximize detection of Type IV SNPs
(A/T). Furthermore, KAPA HRM FAST uses a specialized DNA
polymerase that was engineered for fast amplification, saving time
and increasing throughput. KAPA HRM FAST is a highly sensitive
and reliable product for performing HRM assays.
Amplification and HRM using KAPA HRM FAST. KAPA HRM
FAST Master Mix is designed for high performance detection of
DNA sequence variations. The kit contains a novel DNA polymerase,
engineered via a process of molecular evolution, for fast and
efficient DNA amplification in the presence of high concentrations
of intercalating fluorescent dyes. This allows for rapid amplification
and maximum discrimination between sequence variants. Typical
reaction setup and cycling protocol is shown in Table 3 and 4.
KAPA HRM FAST Master Mix does not contain MgCl2. A separate
vial of 25 mM MgCl2 is supplied to enable easy MgCl2 optimization.
Pilot experiments involving the titration of MgCl2 at 1.5 mM, 2.5 mM
and 3.5 mM are recommended, with further refinement if required.
Amplification of the target DNA is performed using standard
PCR techniques. To maximize throughput of samples, amplification
may be performed on a different instrument before transfer to an
HRM-capable instrument for analysis. KAPA HRM FAST PCR
Kits are stable for up to 1 year if stored properly at -20 C. Once a
reaction has been set up, the EvaGreen dye in KAPA HRM FAST
is stable for ~24 hours, provided the samples are kept at 4 C and
protected from light. Typically, the HRM is performed immediately
after the amplification step is complete. A post-amplification melt at
95 C for 10 60 sec will help the annealing of heterozygote samples
and ensure all amplicons are properly melted prior to HRM.

Component

Recommended
Volume1

Final
Concentration

PCR grade water

Up to 20 l

KAPA HRM FAST

Master Mix (2X)

10 l

1X

MgCl2 (25 mM)2

2 l

2.5 mM

Forward Primer3
(10 M)

0.4 l

0.2 M

Reverse Primer3
(10 M)

0.4 l

0.2 M

Template DNA

1 l

100 pg - 20 ng
(gDNA)

1
Reaction volumes may be scaled down from 20 l to 10 l if low volume tubes/plates
are supported by the instrument.

2
Use 2.5 mM final concentration of MgCl2 as a starting point. A final concentration of
1.5 mM - 4.0 mM is recommended for most assays. Refer to page 9 for more information
on optimizing MgCl2 for HRM.

Use 0.2 M of each primer as a starting point. A final concentration of 0.1 M - 0.5 M
of each primer is recommended for most assays. Refer to page 8 for more information
on optimizing primer concentrations for HRM.

Table 4

Elimination of amplicon contamination prior to assay. In

an uncontrolled environment, it is relatively easy to contaminate
reactions with amplicons from a previous run. Since HRM is very
sensitive, amplicon contamination will result in the mis-calling
of sequence variations, and manifest as mixed or heterozygote
samples. For this reason, KAPA HRM FAST includes dUTP instead
of dTTP. The inlcusion of dUTP facilitates digestion of any amplicon
product with Uracil DNA Glycosylase (UDG) prior to PCR, which
effectively eliminates amplicon contamination. UDG is not provided
in the kit and must be supplied by the end user.

Step

Temperature

Duration

Cycle

Enzyme
activation

95 C

3 min

Hold

Denaturation

95 C

5 sec

Annealing/
Extension

60 C

20 - 30
sec1

Denaturation

95 C

10 - 60
sec

Hold

HRM2

60 - 90 C

35 - 45

The annealing/extension time will vary depending on the ramp speed of the
instrument. For instruments with a slow ramp speed, a shorter annealing/extension
times can be used. For longer amplicons (>150 bp), the addition of an extra
10 sec/100 bp may be required.
1

HRM settings are instrument-dependent.

15

Application Guide
High Resolution Melt Analysis

Troubleshooting
The problems encountered when performing a HRM assay are usually easily identified. The table below details the most common problems
encountered in the PCR and HRM steps, and summarizes previously discussed abnormal data and additional aberrant results. Other
resources are available on the Kapa Biosystems website (www.kapabiosystems.com).

Symptom

PCR

Amplification of the
majority of samples
have a Ct > 30, resulting in the reaction not
reaching plateau.

Ct of samples are
inconsistent.

Non-specific
amplification

Multiple melting
peaks appear for one
product.

Replicate samples
show a wide variance
during HRM analysis
(i.e. results do not
allow accurate genotyping, particularly between homozygotes).

HRM

Only one homozygote

is detected during
HRM.

Different genotypes
cannot be detected
during HRM.

Possible Cause

Solution

Low template concentration

Increase concentration of template DNA.

Poor priming efficiency

Reduce annealing temperature.

Increase annealing time.

MgCl2 concentration is not optimal

Optimize the MgCl2 concentration (e.g. attempt 1.5 mM, 2.5 mM, 3.5 mM MgCl2).

DNA is damaged

Re-purify the DNA.

Varying DNA concentrations

Re-check the DNA concentrations.

DNA is damaged

Re-purify the DNA.

Mispriming

Increase the annealing temperature during amplification/extension.

Check for mispriming using a software package and redesign the primers if necessary.
Reduce primer concentration to minimize primer-dimers and non-specific priming.

MgCl2 concentration is not optimal for primer set

Optimize the MgCl2 concentration (e.g. attempt 1.5 mM, 2.5 mM, 3.5 mM MgCl2) for amplification.

Two products of the same length

Check the products on an agarose gel.

Heterozygosity can result in two peaks.

A HRM melt uses more readings than a standard

melt that can give the impression of 2 products
on a melting peak

Check the products on an agarose gel.

Adjust the filter settings of the melt peak analysis to minimize noise.
Increase the annealing/extension temperature to help minimize non-specific amplification.

Variations in reaction mixture (e.g. salt)

Check the purity of the template solution.

Dilute all template samples in the same buffer (10 mM Tris-HCl, ph 8.5) to equalize salt content.
Re-purify the DNA using a new method.
Ensure all reagents and Master Mixes are vortexed well prior to reaction setup.

Concentrations of template are very different

Ensure all samples are the same DNA concentration for maximum resolution.

Rare or new generic variants may generate

results outside of the expected ranges

Sequence the unusual results.

Redesign primers to produce a shorter amplicon, hopefully avoiding the genetic variation.
Mixing the amplicons (9.5 l from each of two amplicons) can help identify potential new variants.

Secondary structure in the amplicon

Use a Melting Profile software system to ensure no secondary structure are present (eg. http://mfold.rna.
albany.edu/?q=DINAMelt).

Difficult to detect mutation (typically Type IV, A/T)

May need to sequence to confirm, or use a probe or snapback primers.

Only one homozygote is present

Mix 9.5 l of each of the homozygote controls and run a HRM analysis (include an initial melt at 95 C for
3 min); the melting profile should change to the heterozygote, which is usually easier to detect.

MgCl2 concentration not optimal

Perform amplification and HRM at 1.5, 2.5, 3.5 mM MgCl2 to find the optimal concentration for HRM
analysis.

Non-specific amplification

Check the melting curve to see if multiple peaks are present (adjust filter settings if necessary).
Run product on a 2 - 3% agarose gel to confirm if multiple bands are present.
Reduce primer concentration to minimize primer-dimers and non-specific priming.
Increase the annealing/extension temperature.
Redesign primers.

Template contains HRM inhibitors (eg. salt)

Re-purify DNA template.

Difficult to detect mutation (typically Type IV, AT)

Shorter amplicons are better for detecting Type III and Type IV mutations.
Redesign the primers to produce a smaller amplicon.
Use a probe or snap-back primers.

Multiple melt regions or secondary structure

Presence of mutation(s) can result in more complex melting curves due to the development
of secondary structures. This can still provide reliable HRM data.
Long amplicons are more likely to develop secondary structures.
Redesign primers to amplify a shorter fragment.

Non-specific amplification

Increase the melting/annealing temperature during amplification.

Optimize the MgCl2 concentration (e.g. 1.5 mM, 2.5 mM, 3.5 mM MgCl2)

Unusual appearance
of melt curves.

16

Application Guide
High Resolution Melt Analysis

Ordering information
Please note that KAPA HRM FAST PCR Kits are not recommended for quantitative PCR (qPCR). KAPA SYBR FAST qPCR Kits and KAPA
PROBE FAST qPCR Kits are the recommended kits for qPCR. KAPA Express Extract Kits can be used for fast DNA extraction prior to
amplification and HRM analysis using KAPA HRM FAST PCR Kits.

Description

Code

Kit contents

KAPA HRM FAST PCR Kit

KK4201

100 reactions

KAPA HRM FAST PCR Kit

KK4202

500 reactions

KAPA HRM FAST PCR Kit

KK4203

1000 reactions

KAPA Express Extract

KK7100

50 reactions

KAPA Express Extract

KK7101

100 reactions

KAPA Express Extract

KK7102

250 reactions

KAPA Express Extract

KK7103

500 reactions

For more information please contact sales@kapabiosystems.com or your local representative.

Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchasers own internal research. No other patent rights are conveyed
expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California
94404, USA.
EvaGreen is a trademark of Biotium, Inc. Kapa Biosystems is licensed by Biotium, Inc to sell reagents containing EvaGreen dye for use in HRM, for research purposes only.
The purchase of this product includes a limited, non-transferable license under U.S. Patent No. 5,871,908, owned by Evotec Biosystems GmbH and licensed to Roche Diagnostics GmbH, to use only the enclosed
amount of product according to the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent No. 5,871,908, other than
for the amount of product contained herein.
Licensed under U.S. Patent Nos. 5,338,671 and 5,587,287 and corresponding patents in other countries.

Boston, Massachusetts, United States

600 West Cummings Park, Suite 2250
Woburn, MA 01801 U.S.A.
Tel: +1 781 497 2933 Fax: +1 781 497 2934
Cape Town, South Africa
2nd Floor, Old Warehouse Building, Black River Park,
Fir Road, Observatory 7925, Cape Town, South Africa
Tel: +27 21 448 8200 Fax: +27 21 448 6503

www.kapabiosystems.com

Email: info@kapabiosystems.com
17