Application note

Analysis of DNA melting through UV-Visible

absorption spectroscopy
Effect of base-pair mismatch and solvent environment

Introduction
A B
Deoxyribose nucleic acid (DNA) is a naturally occurring
polymer made up of four unique nucleic acid subunits (thymine,
adenine, cytosine, and guanine) referred to as “bases.” When
connected through a phosphate sugar backbone, a single DNA
strand is formed. The bases which make up single-stranded
DNA (ssDNA) can associate or “pair” with bases on a separate
ssDNA sequence through hydrogen-bonding interactions. The Figure 2: Depiction of the formation of single stranded DNA (a) and a
general energy diagram for the DNA melting process (b) where Ea is the
most common pairing structure, Watson-Crick, consists of activation energy of the system.
two hydrogen bonds between thymine and adenine and three
hydrogen bonds between cytosine and guanine,1 as shown in DNA melting, or denaturation, describes the process
Figure 1. Once paired in this manner, the newly formed double- under which dsDNA disassociates into its single-stranded
stranded DNA (dsDNA) forms the well-characterized double- components (Figure 2a and equation 1).
helix secondary structure.
dsDNA ↔ 2ssDNA (1)

A B
As described previously, dsDNA is held together through
hydrogen bonding interactions between the base pairs within
the sequence (Figure 1). To form ssDNA, these interactions
must be broken, and the system must exist in conditions which
thermodynamically favor ssDNA over ddDNA. Considering
thermodynamics, an energy activation barrier must be
overcome to push the system towards the formation of ssDNA
Figure 1: Hydrogen bonding interactions between (a) adenine and thymine
from dsDNA (Figure 2b). The energy needed to overcome this
and (b) cytosine and guanine Watson-Crick base pairs. barrier is specific to the composition of the DNA sequence,
DNA serves as a template by which other materials are and as such, can serve as a useful analysis method for probing
derived and has been used in a myriad of pharmaceutical DNA structure. The transition from dsDNA to ssDNA can
applications, including gene therapy and vaccine development, occur when the system is elevated to higher temperatures. By
among others. 2–5
In these uses, the makeup of the sequence introducing heat, the energy of the system increases, providing
is integral for the synthesized material to perform as intended, the energy needed to transition from dsDNA to ssDNA.7 By
requiring analysis and confirmation of the overall composition. convention, the melting temperature (Tm) is defined as the
DNA melting experiments can be particularly useful for temperature at which half of the dsDNA has denatured.1,8
understanding the structure of the material and has previously
been used to study gene expression.6
DNA has previously been well characterized through will also impact the melting temperature, as well as mutations
spectroscopic methods and is known to absorb in the UV within the structure which result in no pairing between bases
spectral region, with an absorption maximum of 260 nm.9 This (ex: base-pair mismatch).13
absorption feature comes from π → π* transitions related to the
Beyond structural effects, the solvent environment, including
p-orbitals on the purine or pyrimidine aromatic ring.8,9 When
added compounds, can also influence the melting temperature
in the double helix structure, the hydrogen bonds disrupt the
and must be considered when performing melting experiments.
resonance structure of each base, decreasing the strength of
These effects include the effect of buffer conditions/salt
the π → π* transition. This weaker transition results in a smaller
concentration, which can stabilize the dsDNA secondary
extinction coefficient (ε) for the overall structure. From Beer’s law,
structure, requiring a higher temperature to effectively denature
A = clε (2) the material.14,15 Compounds in solution which can bind to
DNA, such as fluorescent probes, can also change the melting
where A is the measured absorbance, c is the concentration
temperature.16 Consequently, it is important to consider not only
of the analyte, and l is the path length, a smaller ε will result
the makeup of the DNA sequence itself, but the interactions the
in a lower observed absorbance for the dsDNA compared to
DNA can have with various components present in the solvent
the ssDNA. Consequently, by monitoring the observed change
as well when analyzing melting temperature.
in absorption at 260 nm as a function of temperature, we can
observe the point at which half of the dsDNA has disassociated Herein, the melting temperature for both small (40 base pairs
into its single-stranded components. As UV-Visible absorption per strand) and large (~2,000 base pairs per strand; calf thymus)
spectroscopy is a non-destructive technique, this analysis can be dsDNA sequences was determined. Absorption measurements
carried out using samples which can be further analyzed later. were obtained using a Thermo Scientific™ Evolution™ One Plus
UV-Visible Spectrophotometer equipped with the Quantum
In an ideal scenario, the resulting sigmoidal curve, as shown in
Northwest t2 Sport Peltier cuvette holder to change the
Figure 3, should be observed, where no change is observed
temperature of the sample. The melting temperature was
at low temperatures (where dsDNA is favored), a steep change
calculated within the Thermo Scientific™ Insight™ Pro Software.
in absorption in the melting temperature region and a second
As described earlier, the melting temperature is sensitive to a
region where absorbance is constant (where ssDNA is favored).
variety of factors, including changes to the solvent environment
The inflection point of this curve will correlate to the melting
and DNA structure. Consequently, additional experiments were
temperature of the DNA sample.
performed to demonstrate the effect of base-pair mismatch, dye
binding, and salt concentrations on the melting temperature.

Experimental
Materials and sample preparation
Four 40 base-pair long DNA strands were purchased through
Integrated DNA Technologies and are described in Figure 4.
Seq. 1–3 are nearly identical; however, 3 bases were changed for
Seq. 2 and 6 were changed for Seq. 3. Each ssDNA sample was
reconstituted with tris-EDTA (TE) buffer (1X, pH 7.4) to achieve a
concentration of 10 ng/μL. Equivalent proportions of the

Figure 3: Ideal DNA melt curve. AdsDNA corresponds to the absorption due
to dsDNA, and A ssDNA refers to the absorption due to ssDNA. Tm is the
melting temperature, defined as the point at which half of the dsDNA is in
the single-stranded form.

As described previously, melting temperature is specific to the

sequence of DNA. For example, the amount of GC base pairs
present in the double-stranded structure will inherently require
more energy, and consequently a higher melting temperature.10
This arises as GC pairs involve three hydrogen bonds as Figure 4: 40 base-pair ssDNA sequences.
opposed to the two hydrogen bonds needed for AT pairs
appropriate sequence (Seq. 1, 2, or 3) and the complement
(Figure 1). The nearest neighbor bases, which are “stacked”
were then mixed together and diluted with TE to ensure the
with bases above and below in the strand, can also influence
same concentration of each strand was present in the solution.
the melting thermodynamics through additional stabilizing
A total of three dsDNA solutions were made, each using the
interactions, influencing the melting temperature.9,11 Additionally,
same complement strand with different sequences (Seq. 1–3).
any non-traditional secondary structure, including i-motif,12
The samples were then annealed by raising the temperature Instrumentation and experiment parameters
of the solution to 90 ˚C, and then cooling back down to room For all absorption experiments, the Evolution One Plus UV-Visible
temperature (~25 ˚C), ensuring the sequences were able to form Spectrophotometer was used to collect the absorption of each
the typical double helix structure. For the melting experiments sample at 260 nm. The Quantum Northwest t2 Sport Peltier
performed using an absorption spectrophotometer, each solution accessory was used to control the temperature of the sample.
was diluted again with TE to a concentration of 3.8 ng/μL. The bandwidth was set to 1.0 nm, a 1.0 s integration time, and
a 1.0 s dwell time was used. The temperature ramp was set to
For fluorescence measurements, the SYBR® Select Master
three different stages as described in Table 2. These temperature
Mix (2X), referred to as SYBR Green in the text, was used as
ramps were set such that more points could be collected in the
a fluorescent probe. For each sample sequence, 40 μL of the
range spanning the anticipated melting region. The final stage
SYBR Select Master Mix (2X) was added to 20 μL of 10 ng/μL
was used to cool the solution back down to room temperature.
of the appropriate dsDNA sample. Each sample was further
diluted using 20 μL of TE buffer. The samples were then split All temperature measurements were collected using a temperature
into three replicate samples, each 20 μL in total volume and probe and read through Insight Pro Software. The probe was
loaded into a 96 well plate. suspended in the solution using a Teflon® cuvette cap, preventing
the probe from touching the cuvette walls and limiting the loss
Calf thymus DNA samples were made using Invitrogen™
of solvent from evaporation. The use of a temperature probe is
UltraPure™ Calf Thymus DNA solution (Lot # 2411666). A
vital as the heat from the Peltier accessory will not transfer to the
200 ng/μL stock solution was made by diluting the calf thymus
sample instantaneously. Additionally, all absorption measurements
solution with TE buffer. The NaCl stock solution was made by
were collected while the solution was stirred using a small stir
dissolving 0.29 g NaCl in 10 mL of TE buffer solution (500 mM
bar. This aids in preventing bubbles from sticking to the sides of
NaCl). Two separate NaCl solutions were then made with
the cuvette, which can influence the measured absorbance, and
concentrations of 5 mM and 10 mM.
ensures the heat is dispersed throughout the cuvette evenly.
For the melting experiments, the control sample was prepared
For fluorescence measurements, an Applied Biosystems™
by diluting 100 μL of 200 ng/μL calf thymus with 1.9 mL of TE
QuantStudio™ 6 Pro PCR system was used. Each sample was
buffer to achieve a final concentration of 10 ng/μL. Two more
run in triplicate and held in the appropriate 96 well plate, capable
10 ng/μL calf thymus samples were made using the 200 ng/μL
of allowing UV light to pass through. The samples were then
calf thymus stock solution, however they were instead diluted
heated to 95 ˚C within 15 seconds, cooled back down to 40 ˚C
using the NaCl + TE buffer solutions as described in Table 1.
for 1 min and finally heated back up to 95 ˚C for 5 seconds.
Fluorescence measurements were collected every 0.3 ˚C.

Thermo Scientific Evolution Spectrophotometers

Table 1: Sample preparation concentrations and amounts for calf thymus with and without NaCl present.

Buffer solution
Volume of 200 ng/μL [NaCl] in sample
Sample [NaCl] in TE buffer Volume of buffer
calf thymus (μL) (mM)
(mM) added (mL)
1 100 0.0 1.9 0.0
2 100 5.0 1.9 4.8
3 100 10.0 1.9 9.5

Table 2: Temperature ramp parameters for melting experiments performed using the Evolution One Plus Spectrophotometer equipped with the
Quantum Northwest Peltier accessory.

Measurement
Final temperature Ramp rate Hold time
Stage interval
(˚c) (˚c/second) (seconds)
(seconds)
1 40 0.0833 2 30
2 95 0.0167 15 30
3 25 0.0833 0 N/A
Melting temperature analysis methods All three of the outlined analysis methods are available through
Practically, the melting temperature of an ideal system, as the Insight Pro Software. In the absorption measurements
shown in Figure 3, can be determined through a “horizontal- described herein, either the slope intercept or inflection method
intercept” method. In this analysis, the dsDNA and ssDNA was used as the collected melt curves did not display ideal
temperature regions are fit to two separate linear functions behavior. For the fluorescence measurements, the melting
which do not depend on the melting temperature, only varying temperature was determined using the inflection method.
in the y-intercept. The average of the two linear functions
for the dsDNA and ssDNA regions is then determined and Results and discussion
the intersection between this average linear function and the Effect of base-pair mismatch
melt curve is taken. This point corresponds to the melting As described previously, melting temperature is highly
temperature of the sample. dependent on the makeup of the DNA sequence, including the
presence of mutations. This sensitivity to deviations from the
For most DNA samples, the absorbance will likely vary outside of
anticipated sequence makes this analysis a useful check prior
the melting region, preventing the use of the horizontal intercept
to further processing and scale-up. To highlight this principle,
analysis method. Instead, two alternative methods can be used:
the melting temperature was determined for three dsDNA
the “slope-intercept” and “inflection” methods. In the slope-
sequences consisting of 40 bases per strand with varying
intercept analysis, similar to the horizontal intercept analysis, the
amounts of mismatched bases (Figure 5). Melting temperatures
temperature ranges before and after the melting temperature
were determined using the slope-intercept method described
regime are fit to two different linear functions with dependence on
previously and are reported in Table 3.
the temperature (y=mx+b). Typically, the slope of these functions
will be the same. The average of the two functions is then The control DNA sample with no base-pair mismatches was found
determined and the intersection of the average linear function and to have a melting temperature of 58.4 ˚C (Figure 5b). By introducing
the melt curve is reported as the melting temperature. only 3 mismatches to one end of the 40 base-pair DNA structure
(Figure 5c), the melting temperature was lowered by ~6 ˚C.
However, for DNA melt curves which deviate greatly from
When 3 additional base-pair mismatches were included on the
ideal behavior or contain multiple melting temperature ranges
opposite end of the DNA (Figure 5d), the melting temperature was
which overlap, the inflection method is most appropriate. In
lowered again. For the DNA sample with 6 base-pair mismatches,
this method, the first derivative of the melt curve is obtained.
the melting temperature was found to be ~14 ˚C lower than the
Mathematically, the inflection point of a function will be the
control sample. With the loss of traditional base pairing, these
maximum of the first derivative of the initial function. As the
mismatched regions are not expected to be held together through
inflection point of the melt curve correlates to the melting
hydrogen bonding, further lowering the energy needed to denature
temperature, the maximum of the first derivative can be used to
and, therefore, the temperature needed to form stable ssDNA
easily find the melting temperature(s) in a more complex system.
in solution. Consequently, with more mismatches, the melting
temperature should lower, as is observed in Figure 5.

B C D

Figure 5: (a) DNA sequences with and without base-pair mismatches. Absorption vs temperature melting curves for 40 base-pair DNA sequences
(3.8 ng/mL in TE buffer) with (b) 0, (c) 3 and (d) 6 base-pair mismatches. Vertical lines correspond to the calculated melting temperature. Melting
temperatures were determined using the sloped intercept analysis.
Effect of bound compounds sequence of interest. However, as the UV-Visible absorption
Absorption is not the only experimental method by which the method only relies on the DNA itself and not the introduction
melting temperature of a dsDNA sequence can be determined. of an additional probe/bound compound, this technique will
Alternatively, fluorescence spectroscopy can be employed. provide a better measurement of the melting temperature.
In this analysis, a fluorescence dye that can interact with the
Effect of salt concentration
dsDNA is added to the sample. For example, SYBR Green
(Figure 6b) is known to bind to the minor groove of dsDNA.17 A B
When bound, a fluorescence signal is observed. Once the DNA
denatures, SYBR green is no longer bound to dsDNA and will
not be fluorescence, resulting in a similar melting curve as seen
for absorption-based melting temperature measurements.

A B

Figure 7: (a) Absorption vs. temperature melting curves for 10 ng/μL calf
thymus DNA suspended in TE buffer and varying concentrations of NaCl
(black—0 mM NaCl, blue—4.8 mM NaCl, and red—9.5 mM NaCl). Vertical lines
denote the calculated melting temperatures (Tm) for each sample included. (b)
Depictions of the effect of salt concentration on strand repulsion.

While fluorescent probes influence melting temperature

through binding to the minor groove of the DNA double
Figure 6: (a) Fluorescence vs. temperature melting curves of three
40 base-pair DNA sequences (black—0 base-pair mismatch, red—3 base- helix, the presence of ions in solution can also influence the
pair mismatch, and blue—6 base-pair mismatch) each coupled with SYBR
measured melting temperature. Figure 7 includes the melting
green as a fluorescent probe. (b) SYBR Green I structure.
curves collected for three separate calf thymus DNA samples.
This fluorescence method was used to analyze the melting Melting temperatures were determined through the inflection
temperatures of the three 40 base-pair DNA sequences previously method. The three samples vary in concentration of excess
studied using UV-Visible absorption (Figure 6a). The melting salt (0 mM, 4.8 mM, and 9.5 mM NaCl). As shown, increasing
temperature was determined through the inflection method. As the concentration of NaCl results in an increase in the melting
shown in Figure 5, the same trend in melting temperature was temperature of dsDNA from 66.8 ˚C with no NaCl added to 75.0
observed in which dsDNA with more base-pair mismatches ˚C with 10 mM NaCl present in the solution (Table 4).
demonstrated a lower melting temperature compared to the
In this example, the effect the presence of salt has on the
control. However, as shown in Table 3, the melting temperature
backbone must be considered. The phosphate group on the
determined through this fluorescence analysis is between
sugar backbone provides the net negative charge for ssDNA.
12–14 ˚C higher than what was observed through the UV-Visible
Though the hydrogen bonding interactions are strong enough
absorption method. This discrepancy in melting temperature is
to hold dsDNA in the double helix structure, the close proximity
not a result of the technique used but is instead a result of the
of the two negatively charged DNA strands will result in some
presence of SYBR Green in the sample solution.
repulsion. Consequently, this behavior will push the equilibrium
Table 3: Calculated melting temperatures for three separate 40 base-pair towards the formation of ssDNA, which in turn lowers the
dsDNA samples using both absorption and fluorescence-based methods
where the latter used SYBR Green as a fluorescent probe.
energy and temperature needed to denature the dsDNA.

Number of Tm (˚C) Table 4: Melting temperatures for calf thymus DNA with varying
base-pair Absorption Fluorescent concentrations of NaCl.
mismatches method probe method DNA sample [NaCl] (mM) Tm (˚C)
0 (control) 58.4 72.5 0.0 66.8
3 52.1 65.5 Calf thymus 4.8 71.6
6 44.1 56.9 9.5 75.0

When positively charged ions, like Na+, are present in solution,

As SYBR Green is bound to the dsDNA in solution, it serves
they can associate with the negatively charged phosphate
almost as a linker between the dye and the dsDNA. The
groups on the DNA backbone providing stabilization of the
interactions which hold SYBR Green to the minor groove of the
dsDNA structure. These ions will effectively screen the charge
DNA must also be broken before the DNA can denature into
of the two DNA strands, thereby lowering the repulsive forces
single strands. The raised melting temperature reflects the extra
between them. As the repulsion is lowered, the energy barrier
energy needed to effectively separate the two strands from one
for forming ssDNA will rise, requiring a higher temperature to
another in the presence of a bound dye. If using the fluorescent
denature dsDNA. If the concentration of positively charged
probe method, it is important this binding interaction be
ions present is increased, the repulsive forces will continue to
considered when estimating the melting temperature of the DNA
reduce until there are enough positively charged ions added
to produce a net neutral charge. As buffers, like tris-EDTA, References
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