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ISSN: 0738-8551 (print), 1549-7801 (electronic)

Crit Rev Biotechnol, Early Online: 1–12

! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.993589

REVIEW ARTICLE

Prediction of melting temperatures in fluorescence in situ hybridization

(FISH) procedures using thermodynamic models
Sı́lvia Fontenete1,2,3,4*, Nuno Guimarães1,2,3*, Jesper Wengel3, and Nuno Filipe Azevedo1
1
Department of Chemical Engineering, Faculty of Engineering, LEPABE, University of Porto, Porto, Portugal, 2Institute of Molecular Pathology and
Immunology of the University of Porto, Porto, Portugal, 3Department of Physics, Chemistry and Pharmacy, Nucleic Acid Center, University of
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Southern Denmark, Odense M, Denmark, and 4ICBAS, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal

Abstract Keywords
The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of DNA, locked nucleic acids, nucleic acid
one, two or more complementary nucleic acid strands, has been studied for many years. The mimics, peptide nucleic acids
appearance of the nearest-neighbor model led to several theoretical and experimental papers
on DNA thermodynamics that provide reasonably accurate thermodynamic information History
on nucleic acid duplexes and allow estimation of the melting temperature. Because there
are no thermodynamic models specifically developed to predict the hybridization temperature Received 24 October 2013
of a probe used in a fluorescence in situ hybridization (FISH) procedure, the melting Revised 28 October 2014
temperature is used as a reference, together with corrections for certain compounds that are Accepted 28 October 2014
used during FISH. However, the quantitative relation between melting and experimental FISH Published online 14 January 2015
temperatures is poorly described. In this review, various models used to predict the melting
For personal use only.

temperature for rRNA targets, for DNA oligonucleotides and for nucleic acid mimics (chemically
modified oligonucleotides), will be addressed in detail, together with a critical assessment of
how this information should be used in FISH.

General concepts is placed in contact with the target cells, and if complemen-
tary (or near-complementary) sequences are present, hybrid-
Fluorescence in situ hybridization (FISH) is a powerful
ization will take place. The specificity of this binding event,
molecular method with widespread use in environmental and
i.e. the ability of the method to discriminate the target
in medical applications for the identification, visualization,
organisms from the remaining cells, is further ensured by a
and quantification of organisms of interest in microbial
washing step where all loosely-bound probes are washed
communities (Amann et al., 1990, 1995; Amann & Fuchs,
away. Specificity can be theoretically predicted using 16S
2008; DeLong et al., 1989; Guimaraes et al., 2007; Wagner
rRNA comparative sequence analysis, as probes are designed
et al., 2003). The FISH procedure is based on the detection of
to confer a required level of taxonomic specificity (e.g.
nucleic acids sequences by a fluorescently labelled nucleic
species, genus, class), and then implemented in the laboratory
acid probe that hybridizes specifically to complementary
by optimizing the hybridization and washing conditions
target sequences within intact microbial and animal cells
(Hugenholtz et al., 2002). Finally, visualization by either
(Moter & Gobel, 2000). It involves five major steps: fixation,
fluorescence microscopy or flow cytometry allows the
permeabilization, hybridization, washing and visualization.
researcher to observe if successful hybridization has occurred.
Fixation and permeabilization is typically joined in one
A successful hybridization is dependent on several factors
operation with the objective to render the cell wall permeable
such as the conditions at which the hybridization step is
to the nucleic acid probe entry, while, at the same time,
carried out and the ribosomal content of cells (Behrens et al.,
guaranteeing that cell lysis and extensive nucleic acid
2003). The effect of ribosomal proteins, the affinity of the
degradation will not occur. During hybridization, the probe
probe and technical factors like the type of fluorophore used,
and the microscope’s optical quality are also very important
(Amann et al., 1995; Behrens et al., 2003; Hoshino et al.,
2008; Yilmaz & Noguera, 2004). Due to this large number of
factors involved, the development of new FISH procedures
*These authors contributed equally to this work.
remains highly empirical and time-consuming. One of the
Address for correspondence: Nuno Filipe Azevedo, Department of ways to decrease the effort associated with method develop-
Chemical Engineering, Faculty of Engineering, LEPABE, University of
Porto, rua Dr. Roberto Frias, Porto 4200-465, Portugal. Tel: +351 22 508 ment is to be able to predict under what conditions a new
1652. E-mail: nazevedo@fe.up.pt probe will work. For instance, if a suitable mathematical
 2 S. Fontenete et al.
model is available that allows the researcher to predict
the temperature at which the hybridization step should be
carried out, the need for trial-and-error experiments
where several temperatures are tested can be avoided or at
least minimized.
When FISH first appeared, most studies were performed
at the same temperature (46  C) during the hybridization
step (Manz et al., 1995; Neef et al., 1995; Wagner et al.,
1994). This temperature was maintained constant by testing
different probe designs and altering the concentration of a
denaturing agent, such as formamide, in the hybridization
solution. The arrival of multiplex methods (the use of more
than one probe in the same experiment to detect multiple
microorganisms), together with a better understanding of the
hybridization mechanisms, changed this approach. Because
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in a multiplex experiment probes have to operate at the same
temperature and concentrations of denaturing agents, now
researchers tend to try and predict the temperature at which
the probes will work based on the sequence of the probe and
taking into account the compounds of the hybridization
solution used in the experiment (Cerqueira et al., 2008;
Fontenete et al., 2013; Kubota et al., 2006). However, to date,
there are no mathematical models that can accurately predict
the optimal hybridization temperature (TH) for a given
oligonucleotide probe. As such, the more easily calculated
melting or mid-transition temperature (Tm) of an oligonucleo-
tide is calculated instead (SantaLucia & Hicks, 2004). Tm can
For personal use only.
be defined as the temperature at which half of the nucleic-acid
strands are forming a duplex and the other half are single
stranded, i.e. the temperature at which the dissociation factor
is 0.5 (SantaLucia et al., 1996). Typically, Tm is used as an
indication for the hybridization temperature, but the relation-
ship between these temperatures has not been clearly defined
in the literature. It appears sensible to state, however, that for
hybridization to occur as efficiently as possible, it would be
desirable that all targets sites have bound probes. It hence
follows that Tm overestimates TH by a value that is related
to the slope of the melting curve and to the dissociation
fraction that is considered acceptable for the FISH procedure
(Figure 1). For strands that are not completely complemen-
tary, i.e. that contain one or more mismatches to the target
sequence, it is usually desirable that the dissociation factor is
close to 1 at the hybridization temperature.
Examining Figure 1, it would be reasonable to consider
that the hybridization would be successful if it was performed
at a temperature equal or lower than the TH. However, in the
initial state of the dissociation fraction curves, the nucleic
acids are already in the duplex configuration, and as such do
not require energy – so called activation energy (Ea) – to form
a duplex. FISH, on the other hand, starts with two single
stranded strands that will require Ea to hybridize (Yilmaz &
Noguera, 2007). This implies that low temperatures will make
the hybridization process very slow, and consequently there is
a narrow range of TH at which FISH will provide satisfactory
results. This narrow range allows researchers to increase the
temperature to minimize non-target hybridization, if specifi-
city is an issue, whereas the opposite may be true if sensitivity
is the biggest issue.
Tm is intrinsically related with other thermodynamic
properties such as the Gibbs Free Energy (G). This energy
Crit Rev Biotechnol, Early Online: 1–12
Figure 1. Melting temperature (Tm) and dissociation fraction curves are
showed for completely complementary strands (DNA duplex) and for
strands with one mismatch (DNA duplex mismatch). The hybridization
temperature (TH) is presented as the hybridization temperature for DNA
duplex and TH (mm) represents the hybridization temperature for the
DNA duplex containing one mismatch (DNA duplex (mismatch)). The
values used to design the curves are hypothetical.
is a thermodynamic quantity which can be used as an
indicator of whether a reaction is thermodynamically favor-
able or not. When DG is negative (DG50) the reaction will
be favorable or spontaneous, and the more negative the value
the more favorable is the reaction. An estimation of these
parameters (and also their correlation) can now be obtained
by the nearest-neighbor model (NN), which relies on the
assumption that the stability of a base pair depends on the
identity and orientation of the adjacent base pair (neighbor
base pair) (Bloomfield, 2000; SantaLucia, 1998). While very
useful, the NN model was developed to deal with the
thermodynamics of nucleic acid hybridization in general, and
does not take into account all parameters and chemical
compounds involved in the hybridization step of a FISH
procedure.
Here, we will critically review existing models for
prediction of the Tm value and describe corrections (e.g.
for salt and formamide constituents) that exist to approxi-
mate the theoretical model to the chemical micro-environ-
mental conditions of typical FISH procedures. Most of the
modeling work and FISH procedures have been developed
for DNA probes, and as such we will start by reviewing
models for DNA molecules. Nonetheless, DNA probes in
FISH are being replaced by better performing nucleic acid
mimics, such as peptide nucleic acid (PNA) and locked
nucleic acid (LNA). Consequently, the existing models for
these novel types of mimics and respective corrections will
also be presented. Furthermore, while in the microbiology
area the probe target is mostly a RNA molecule, the existing
Tm models were originally developed for a DNA target.
While some of these models have been adapted to RNA
(Freier et al., 1986; Sugimoto et al., 1995), we decided for
consistency to present first the models targeting DNA and
only afterwards, when available, mention the adaptations for
RNA. The last section of the review compares the predicted
values of Tm against TH obtained in the laboratory and
provides some explanations for observed discrepancies and
suggestions for future work.
 DOI: 10.3109/07388551.2014.993589
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Figure 2. Major milestones in DNA and nucleic acid mimics.
Melting temperature prediction of oligonucleotide
probes
Melting temperature of DNA duplexes and DNA:RNA
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hybrids
The prediction of a precise Tm for DNA duplexes was an early
concern of many researchers (Figure 2). Marmur had already
proposed a simple formula to predict this temperature from
the GC content of a sequence (Tm ¼ 4GC + 2AT) (Marmur &
Doty, 1962). However, this model is simplistic because it only
considers the type and number of nucleic acid bases of a
sequence.
In order to improve from the previous equation, various
studies have reported thermodynamic values for calculating
DNA stability from the order of bases within a sequence
based on the NN model, and several NN parameter sets
for predicting DNA duplex stability are described in the
literature (Breslauer et al., 1986; Gotoh & Tagashira, 1981;
Ornstein & Fresco, 1983). From all these parameter sets, the
NN parameters published by SantaLucia & Hicks (2004)
are the most commonly used for the calculation of thermo-
dynamic values.
The NN model starts by assuming that enthalpy (DH ) and
entropy (DS ) are independent. It is then possible to determine
the DG for nucleic acid duplexes of different lengths as a
sum of 2 base pair duplexes (DGT ). The DGT value
is obtained through Equation (1) using DH and DS values
(T is in Kelvin, DH is in cal mol1 and DS is in units of
cal K1 mol1) (SantaLucia & Hicks, 2004).
DGT ¼ DH  þ TDS ð1Þ
A derivation of Equation (1) can be used to determine
the Tm on the basis of the thermodynamic parameters. In fact,
Tm ( C) may be calculated by Equation (2), where R is the
gas constant 1.9872 cal/K – mol, Ct is the total strand
probe concentration (mM) and x is the symmetry factor
Melting temperatures in FISH 3
(where x is equal to 1 for self-complementary oligonucleotide
duplexes and equal to 4 for non-self-complementary oligo-
nucleotide duplexes) (SantaLucia & Hicks, 2004).
1000
Tm ¼ DH   ð2Þ
½DS þ R  lnðCxt Þ  273:15
The deviation of this model has been estimated to be
2.3  C for DNA duplexes but so far efforts to obtain a better
model have been unsuccessful (Owczarzy et al., 1999;
SantaLucia & Hicks, 2004). This model can also be used
for DNA:RNA duplexes by simply adapting the NN param-
eters for this type of situation with an average error of 0.6%
(Sugimoto et al., 1995).
Melting temperature of PNA:DNA duplexes
PNA is an uncharged synthetic DNA analogue which is
resistant to nuclease and protease degradation. PNA forms
various hybrid complexes such as PNA:PNA, PNA:DNA
and PNA:RNA duplexes, and PNA:PNA:PNA and
PNA:DNA:PNA triplexes (Rasmussen et al., 1997). Due to
its thermodynamic characteristics, PNA presents several
advantages for FISH experiments. For instance, PNA:target
duplexes proved to be more stable under physiological salt
conditions and to have more favorable hybridization proper-
ties than the corresponding unmodified DNA or RNA
duplexes (Egholm et al., 1993; Seo et al., 2011; Sen &
Nielsen, 2006). PNA probes can be shorter than nucleic acid
probes due to their enhanced binding properties (Demidov
et al., 1994), and, because PNA is an uncharged oligonucleo-
tide analogue, it has a higher capacity of penetrating untreated
bacterial cell walls than their negatively charged nucleic acid
counterparts (Good & Nielsen, 1998). Due to its neutral
charge, PNA has very high affinity for DNA/RNA due to
the lack of charge repulsion of the peptide-like backbone
(Egholm et al., 1993; Giesen et al., 1998; Jensen et al., 1997).
 4 S. Fontenete et al.
The higher specificity and sensitivity have been exploited in
order to develop several diagnostic applications (Cerqueira
et al., 2013; Grossmann et al., 2008; Zhang & Appella, 2007).
Several authors have described that the higher Tm of PNA-
containing duplexes depends on the sequence and length of
the duplexes (Chakrabarti & Schwarz, 1999; Giesen et al.,
1998) and generally can be explained by an enthalpy increase
(Chakrabarti & Schwarz, 1999). Theoretical predictions of
the thermodynamic behavior of PNA:DNA duplexes have
employed NN values and empirical thermodynamic param-
eters (Griffin & Smith, 1998). Since these models were
initially built for Tm prediction of DNA:DNA duplexes, not
surprisingly they proved inaccurate when applied to
PNA:DNA duplexes (Griffin & Smith, 1998). Accordingly,
these models are also not applicable to predict accurate
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PNA TH values. It was therefore necessary to determine a
new set of thermodynamic parameters to represent the
average contribution of each PNA base in order to be able
to accurately predict the Tm values for PNA-containing
duplexes (Chakrabarti & Schwarz, 1999).
In another approach, Giesen et al. (1998) developed an
empirical formula based on the Tm value as calculated for the
corresponding DNA:DNA duplex (SantaLucia et al., 1996).
This formula includes terms for the pyrimidine content fpyr
and length of the PNA (Equation 3).
Tmpred ¼ c0 þ c1  Tmn mDNA þ c2  fpyr þ c3  length ð3Þ
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The constants were determined to be c0 ¼ 20.79, c1 ¼ 0.83,
c2 ¼ 26.13 and c3 ¼ 0.44.
However, the thermodynamic values calculated from the
above mentioned models are not nearly as accurate as values
calculated from the DNA duplex models (Griffin & Smith,
1998; Giesen et al., 1998), likely because PNA models have
been much less studied than their DNA counterparts.
Therefore, the development of a more accurate thermo-
dynamic model for PNA is a priority to be able to predict
more accurately not only hybridization reactions of
PNA:DNA duplexes but also PNA:RNA duplexes.
Melting temperature of LNA:DNA duplexes
Locked nucleic acid (LNA) is another nucleic acid mimic that
has recently began to be used in FISH (Fontenete et al., 2013;
Kubota et al., 2006; Robertson & Vora, 2012; Silahtaroglu
et al., 2004). The pentose ring of LNA nucleotides is
conformationally locked in a C30 -endo/N-type sugar conform-
ation by an O20 to C40 methylene linkage (Kumar et al., 1998;
Obika et al., 1997), and has been used as an antisense
molecule both in vitro and in vivo, exhibiting very high
affinity and specificity toward complementary DNA and
RNA (Robertson & Vora, 2012; Vester & Wengel, 2004;
Zhang et al., 2011). The use of LNA residues in oligonucleo-
tides allows the stabilization of the duplex during the
hybridization and permits a more sensitive detection
(Kubota et al., 2006). Additionally, this stability is evident
in biological media where only the presence of three LNAs at
the 50 and 30 ends is enough to increase the half-life of the
oligonucleotide (Kurreck et al., 2002). LNA has important
advantages comparatively to PNA, namely the possibility of
being synthesized using conventional phosphoramidite
Crit Rev Biotechnol, Early Online: 1–12
chemistry which allows automated synthesis of chimeric
oligonucleotides such as DNA + LNA or LNA + RNA.
Additionally, PNA probe synthesis is limited in terms of
length and sequence, which increases the difficulty of
designing PNA probes with enough specificity
(Perry-O’Keefe et al., 2001). For this reason, LNA probes
offer high design flexibility comparatively to the PNA probes.
The introduction of LNA into an oligonucleotide raises the Tm
of the hybrid, depending on the length and the position in the
oligonucleotide. It has been shown that the number of LNA
substitutions needs to be optimized to allow a higher
hybridization efficiency and specificity (Kubota et al., 2006;
Piao et al., 2009). It also been proved that single LNA
modifications in the inner positions of the duplexes has equal
effect on the stability, in spite of the chemical nature of the
LNA nucleotides used (Kaur et al., 2006; Vester & Wengel,
2004). However, the additive behavior of multiple LNA
substitutions was only observed if the LNA nucleotides were
separated at least by one 20 -O-methyl nucleotide (20 OMe
RNA) (Petersen et al., 2002). Similarly, LNA/DNA probes in
which LNA and DNA nucleotides alternate have shown
strong increase of affinity (Kubota et al., 2006). However, the
thermodynamic origin of LNA’s enhanced base pairing
stability has not been clearly delineated, although it has
been suggested that decreased entropy of duplex formation
and improved stacking in the duplex both play a part
(Owczarzy et al., 2011).
Similarly to PNA, LNA parameters differ from the DNA
parameters used in the NN models, but several authors, such
as McTigue et al. (2004) and more recently Owczarzy et al.
(2011), have determined a set of unique LNA parameters that
could be used for prediction of thermodynamic parameters of
the hybridization of LNA oligonucleotides to their all-DNA
complements. McTigue et al. have determined the thermo-
dynamic parameters for the hybridization of DNA oligo-
nucleotides with a single LNA nucleotide to their all-DNA
complements (McTigue et al., 2004). The variation in the
enthalpy parameter due to the LNA nucleotide (DDH  ) was
estimated by the equation below:
DDH   DHLNAþDNA:DNA
 
 DHDNA:DNA ð4Þ
Here LNA + DNA:DNA refers to the duplex with a single
LNA nucleotide incorporated in one strand and DNA:DNA
refers to the reference DNA duplex. The DDS , DDG37 and
DDTM can be obtained by analogous expressions. After
evaluating the accuracy of the experimental parameters in the
test set of oligonucleotides, the authors combined the data of
all the 100 oligonucleotides used and derived a 32 NN
thermodynamic parameter sets for LNA incorporation
(McTigue et al., 2004) (Tables 1 and 2). The 32 NN
parameter set gives a prediction of the Tm of a new single
LNA-substituted sequence. To use it, the DH and DS of the
corresponding all-DNA duplex is calculated using the already
described DNA parameters (SantaLucia et al., 1996). Then
the DDH and DDS values from the 32 NN set for the two
LNA NN pairs are added and the Tm calculated using
Equation (2). According to the authors, the sets of DDH ,
DDS and DDG37 parameters for all the 32 possible NN for
LNA + DNA:DNA hybridization provides Tm estimates
 DOI: 10.3109/07388551.2014.993589
Table 1. Models from the literature for Tm prediction of an oligonucleotide.
Models for Tm prediction
 
Tm ¼ DH =ðDS þ R lnðCA  CB =2ÞÞ
Tm ¼ DH  =ðA þ DSNN þ R lnðCt ÞÞN73:15
Tm ¼ ðDHduplex þ DHnuc Þ=ðR lnðCT Þ þ DSduplex þ DSnuc Þ
DH (cal/mol); mS (cal mol1 K1); Ct (mM)
Tm ¼ DH   1000=ðDS þ R lnðCt =xÞÞ  273:15
DH (kcal/mol); (S (cal mol1 K1); Ct
(mM); R (cal/K-mol)
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Fudan University on 05/07/15
Table 2. Different models for DG prediction existing in the literature for an oligonucleotide.
Models for DG prediction (cal/mol)
DGT 
¼ DH  TDS 
DH (cal/mol);(S (cal mol1 K1);T (K)
DG1 ¼ DHduplex

 TDSduplex
P
DGduplex ¼ ij Nij ðDHij  TDSij Þ
DG1, mismatched ¼ DG1, perfect complementary þ DDG1
DGi ½FA ¼ DGi, 0% þ mi ½FA
0 0 0 0 0 0
3  5 3
DDG1 ¼ DG ð350 cc5  5 3
0 Þ  ðDG ð30 cc50 Þ þ DG ð30 50 ÞÞ
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accurate within 2  C for oligonucleotides containing a single
LNA nucleotide.
Although this study presented important progress on LNA
thermodynamics, the LNA parameter set is incomplete
because most probes have more than just a single LNA
incorporation. For instance, a triplet of LNA nucleotides
appears to maximize mismatch discrimination and improves
single-nucleotide polymorphism assays (You et al., 2006),
genomic DNA sequences can be selectively captured by fully
LNA-modified probes (Jacobsen et al., 2002), and LNA
incorporations induce higher specificities increasing the
mismatch discrimination of FISH probes (Kubota et al.,
2006). In fact, using values determined experimentally and
from previous NN parameters (McTigue et al., 2004;
SantaLucia & Hicks, 2004), Owczarzy et al. establish a new
set of NN parameters that represent the effects on thermo-
dynamic parameters of consecutive LNAs nucleotides (table 1
of reference Owczarzy et al., 2011). Since these values
represent deviations from native DNA duplexes in order to
calculate the total enthalpy of a LNA-modified sequence, one
predicts the transition enthalpy for the native DNA duplex
(DH ) and adds the differential parameters (DDH ) to take
into account the LNA-mediated effects (Equation 5).
bp 1
NX X
LNA
DH  ¼ DHi, iþ1 þ DHinit

þ DDH 
i¼1
To simplify the calculation of thermodynamic values,
Owczarzy et al. (2011) presented a full set of thermodynamic
parameters for consecutive and isolated LNA modifications
(table 2 of reference Owczarzy et al., 2011). Instead of having
Melting temperatures in FISH 5
Units Characteristics Authors/Studies
K Use for non-self-complementary oligonucleotides (SantaLucia, 1998)
duplexes, if the strands are different concentration

C Including all the changes between (Borer et al., 1974)
self-complementary and non-
self-complementary oligomers
K Duplex sequences as a function of strand concentration (Owczarzy et al., 1997)

C Use for self and no self-complementary (SantaLucia & Hicks, 2004)
oligonucleotides duplexes, where x equal 1 or 4
respectively
Characteristics Authors/Studies
Use at a different temperatures (when DH and DS are (SantaLucia & Hicks, 2004)
temperature independent)
For DNA/RNA duplexes (Yilmaz, 2006)
Use for duplex stability (Owczarzy et al., 1997)
When a mismatch form is present. (Yilmaz, 2006)
For probe dissociation with increasing formamide (Yilmaz, 2006)
percentage.
If a mismatch forms a trinucleotide. (Yilmaz & Noguera, 2007)
to calculate the thermodynamics of the native DNA duplex
and then add the differential parameters caused by the LNA
modifications, Owczarzy et al. (2011) combines the param-
eters of an isolated LNA modification (table 4 of ref McTigue
et al., 2004) or consecutive LNA modifications (table 1 of
reference Owczarzy et al., 2011) with the corresponding DNA
NN parameters (table 1 of reference SantaLucia & Hicks,
2004) which gives full NN LNA parameters. The full set of
NN parameters allows a faster calculation of thermodynamic
values involving an LNA-modified sequence. Similarly for
PNA, there are no parameters for LNA:RNA duplexes and as
such it is expected that these models present many deviations
from the reality.
Models currently available to make reaction
parameter corrections that affect the variation of Tm
In Section ‘‘Melting temperature prediction of oligonucleo-
tide probes’’ we have described the more commonly used
models for the theoretical prediction of the melting
temperatures for DNA, PNA and LNA probes. These
models provide an approximation of the possible behavior
of the probe in study, however the results are far from being
precise, partly due to the fact that these models do not
consider the effects of several compounds used in the FISH
ð5Þ
procedure that have crucial influence in the hybridization.
Having this in mind several authors have proposed models to
predict the effect that these parameters have on the Tm
values. As before, these models are mostly developed for
DNA probes and only very limited adaptations for nucleic
acid mimics exist.
 6 S. Fontenete et al.
Models used for hybridization with DNA probes
The denaturing agent formamide (FA) and sodium ions are
two of the most important ingredients used in FISH reactions
with DNA probes, and are also two of the most likely to
impact hybridization temperature.
Effect of formamide on the stability of duplex
FA is used to adjust the stringency of the hybridization. It acts
by allowing a more exposed surface in the molecule, leading
in turn to a thermodynamically more favorable reaction
(Yilmaz & Noguera, 2007). Earlier studies suggested that this
effect of FA was due to its ion-solvating power (Helmkamp &
Ts’O, 1962; Levine et al., 1963) and the ability to increase
the solubility of free bases (Levine et al., 1963). In fact,
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Fudan University on 05/07/15
by increasing the hydrophobic character of the solvent, the
activity coefficients and free energies of the bases were
decreased, favoring the denatured state (Herskovits et al.,
1961; Geiduschek & Herskovits, 1961; Sander et al., 1962).
Initially, it was demonstrated that the Tm of DNA duplexes
decrease linearly by approximately 0.65  C per volume
fraction (100) of FA (Casey & Davidson, 1977; Hutton,
1977; McConaughy et al., 1969; Record, 1967). However,
these earlier studies consider the effects of FA independent of
base content of the DNA.
Consequently, it is necessary to achieve the optimal
denaturing conditions that allow specific hybridization.
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Some studies determined these conditions based on FA
dissociation profiles, which show the variation of brightness
of hybridized cells with the change of FA concentration
(Yilmaz & Noguera, 2007). These FA curves allow the
determination of melting points and can be used to compare
models prediction with experimental results (Wright et al.,
2014). Yilmaz (2006) has developed a FA-based probe
dissociation model where the DG values for each reaction
in the equilibrium model must be obtained for different
FA concentrations. The changes in DG values are obtained
with Equation (6), where the ‘‘m-value’’ is used to formulate
the linear relationship between FA concentrations and the
DG values.
DGi ½FA ¼ DGi, 0% þ mi ½FA ð6Þ
However, this model is independent of the sequence,
although it has been demonstrated previously that the FA
effect is dependent of base content of the DNA (Blake &
Delcourt, 1996).
This model was improved by Wright et al. (2014), through
the use of a retrained mechanistic model (RMM). The RMM
model was developed based on the improvement of the
prediction of FA curves for perfectly matched hybrids, using
new thermodynamic calculations and data sets. These data
sets were calibrated and validated on 106 probes targeting
five different organisms (Wright et al., 2014). Therefore, the
prediction of hybridization efficiency is defined by the ratio
of probe-bound rRNA molecules ([PR]) to total rRNA ([R]0)
(Equation 7).
½PR ½P0 Koverall
¼ ð7Þ
½R0 1 þ ½P0 Koverall
Crit Rev Biotechnol, Early Online: 1–12
This ratio is related with the overall equilibrium constant
(Koverall ) and the molar concentration of probe used ([P]0).
The Koverall constant (Equation 8) is further defined as a
function of three DG changes (i ¼ 1,2,3) representing the
reactions for probe:target duplex formation, probe folding,
and target folding, respectively.
 DG m ½FA
exp  1% RT
1
Koverall ¼ h  DG m2 ½FA
i h  DG m ½FAi
1 þ exp  1% RT 1 þ exp  1% RT
3
ð8Þ
In this equation, R is the ideal gas law constant
(1.99  103 kcal/mol K), and T is the hybridization tempera-
ture in K.
In order to improve predictions of formamide denaturation
in FISH another approach was used from the same authors for
microarrays hybridizations (Yilmaz et al., 2012), the single-
reaction computationally model (SRM) (Wright et al., 2014).
The SRM model converts the DG values from DNA/RNA NN
rules to a specific DG using a linear relationship with only
two parameters, instead of 17, and was considered the best
model in predicting the melting point, with the error being
less than 10%. Nevertheless, because SRM cannot predict
probe affinity, the authors use in parallel RMM to predict
hybridization efficiency as a function of FA (with predicted
DG values and known probe concentration).
The effects of FA in complementary duplexes containing
mismatches are still poorly understood. It is known that an
optimal range of FA concentrations enhance the mismatch
discrimination due to the occurrence of a more rapid
discrimination in duplexes with mismatches (Yilmaz, 2006).
Therefore, the mismatch discrimination in FISH can be
associated with how much the mismatches change the FA
dissociation profile of a probe comparatively to that obtained
with a target microorganism (Yilmaz et al., 2008).
Finally, it is important to mention that several studies
proved that different denaturants could replace FA (e.g. urea,
SDS) (Kubota et al., 2006; Fontenete et al., 2013; Simard
et al., 2001), and have showed that the use of FA is not always
beneficial for the best outcome of the FISH procedure
(Matthiesen & Hansen, 2012).
Effects of mismatches on melting temperature
The appearance of mismatches leads to a less favorable free
energy binding when compared to the original and perfectly
sequence-matched duplex (Ratilainen et al., 2000) with a
decrease in the efficiency of the reaction. This phenomenon is
purely chemical, since the loss of connections and the
surrounding area of the mismatch are matched with a more
rigid structure forming a not so perfect duplex (Ratilainen
et al., 2000). Increasing the size of the probe is usually
performed in an attempt to create a higher affinity with the
target sequence. However, this elongation can increase the
nonspecific binding of the probe, leading to small mis-
matches. Therefore the systematic characterization of mis-
matches stability is an upmost requirement in FISH. However,
this is a complicated process due to several variables
associated with this particular parameter and uncertainties
 DOI: 10.3109/07388551.2014.993589
in the estimation of it. According to the NN model, the ‘‘G’’
base is the one which will have the tendency to form the
highest number of mismatches, while the ‘‘C’’ base has the
most propensity to form very few mismatches (SantaLucia &
Hicks, 2004). Therefore, the stability predictions could help
determining false positives, consequently foreseeing the
published probe specificity.
The quantification of the effect of internal mismatches is
therefore important to understand the cross-hybridization
effect, which could have some practical consequences for
optimal probe design (Hooyberghs et al., 2009). The calcu-
lation of the DG1 , in Equation (9), is essential to determine
the duplex stability.
DG1, mismatched ¼ DG1, perfect complementary þ DDG1
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Fudan University on 05/07/15
The difference in DG1 , referred as DDG1 , represents the
effect of stability mismatch on the duplex DNA:RNA.
Subsequently, the DDG1 needs to be calculated to incorporate
single mismatches into the theoretical framework. The NN
theory suggests that the stability of a mismatch is dependent
of the mismatch type and the base pairs adjacent to the
mismatch (Sugimoto et al., 2000).
The screening of FISH probes for potential non-target
bindings against a large rRNA databases of rRNA sequences
is crucial to reduce the number of candidate probes before
undertake laborious experimental validation process. There
are some programs that can be used to help in this process,
For personal use only.
such as ARB software. ARB, provide a prediction of
mismatch stability, where a weighted mismatch score is
calculated based on the type of mismatch and the relative
perfect match (Ludwig et al., 2004). Yilmaz et al. (2008)
compare the mismatch scores obtained by this software with
the experimental results of the effect of single mismatch on
FA dissociation profiles. Although the authors agree with the
importance of stability predictors to evaluate the specificity of
probes, they reefer that the parameters of ARB should be
improve. Additionally, this software does not consider
insertions and deletions sites, which can outcome false
positive FISH results if the DG of the loop structure is less
than 4 kcal/mol (McIlroy et al., 2011). The software tool
LoopOut allow the screening of FISH probes for non-target
binding containing single nucleotide insertions or deletions.
This tool generates all possible insertion/deletion combin-
ations and the theoretical DG and DGbulge values for each
variant for a FISH probe. DGbulge values were calculated
based on Equations (10) and (11) for pyrimidine and purine
bases, respectively (Znosko et al., 2002).
DGbulge ¼ 3:9 þ 0:1DGnn kcal=mol
DGbulge ¼ 3:3 þ 0:3Gnn kcal=mol
DGnm is the calculated free energy of the neighboring base
pair of the bases flanking the bulge (Znosko et al., 2002).
Several correlations to these models can also be applied
according with the type of insertions/deletions (McIlroy et al.,
2011; Pozhitkov et al., 2006; Znosko et al., 2002).
Even though several approaches have been analyzed to
characterize mismatch stability in FISH, this is a very
complex subject. The use of different microorganisms
Melting temperatures in FISH 7
with mismatches on the target site lead to a different DG
values for the target site, and makes the calculation
uncertain (Yilmaz et al., 2008). Therefore, experimental
and theoretical methodology should be combined to obtain
more accuracy results.
The improvement of the specificity of FISH probes has
been investigated. One of these approaches is the application
of dual probes with distinct fluorophores. These probes
should be design to have similar melting points and a
mathematical modeling study should be determined to
analyse the potential false negative results based on mismatch
thermodynamics (Wright et al., 2014). The increase of
stringency conditions (e.g. higher FA concentration)
(Kirschner et al., 2012; Yilmaz & Noguera, 2007) and the
ð9Þ
use of unlabeled competitor probes (Fuchs et al., 2000;
Kirschner et al., 2012) are other strategies used to minimize
the cross-hybridization with mismatch non-targets and
enhanced the fluorescence signal. However, the use of
stable DNA mimics it is a simpler approach to help to
overleap the mismatch problem. Although several studies
have already found that destabilization occurs in the presence
of mismatches in both DNA:DNA, PNA:DNA and
LNA:DNA duplexes, the ability of discrimination of nucleic
acid mimics is usually higher than when only DNA is used
(Chou et al., 2005; Egholm et al., 1993; Johnson et al., 2004;
Mouritzen et al., 2003).
Effects of sodium on prediction of melting
temperatures
The relationship between sodium ion concentration and Tm of
a DNA duplex has been widely studied with several
algorithms (Owczarzy et al., 1997; SantaLucia, 1998;
SantaLucia et al., 1996), with the algorithm developed by
Owczarzy et al. (2004) being the most accurate. According to
this model, the Tm calculation can be adjusted to the salt
concentration through Equation (12), however DNA concen-
tration and the sequence length are not considered (Owczarzy
et al., 2004).
1 1 ½Naþ 2
¼ þ ð4, 29f ðG:C Þ  3:95Þ  105 ln
TMð2Þ TMð1Þ ½Naþ 1
 
þ 9:40  106 ln2 ½Naþ 2  ln2 ½Naþ 1
ð12Þ
In sum, despite the existence of various models for Tm
value prediction and correction parameters, they were not
ð10Þ developed for FISH, they are not accurate enough for this
specific application. In fact, the model for DNA duplexes
proposed by SantaLucia et al. (SantaLucia & Hicks, 2004;
ð11Þ SantaLucia et al., 1996) is still one of the most used.
Models used for hybridization with PNA probes
The influence of the different parameters in FISH using PNA
probes is well known. Some authors reported the change
in solubility associated with Tm values in duplexes of PNA
(Spink & Chaires, 1999). It thus seems that differences in
hydration of PNA:DNA duplexes can severely influence the
duplexes stability (Sen & Nielsen, 2006).
 8 S. Fontenete et al.
The influence of sodium ion on PNA hybridization has
been controversial. Some authors described that the concen-
tration of sodium ion has an influence on thermodynamic
parameters (Griffin & Smith, 1998) while other studies
reported no significant effects of the ionic strength on
the stabilities of duplexes of PNA (Sen & Nielsen, 2006;
Tomac, 1996).
In particular, the stability of PNA duplexes is dependent
not only on the percentage of GC content but also on the
purine proportion present (Giesen et al., 1998). This occurs
because PNA:DNA duplexes containing a pyrimidine-rich
PNA strand are enthalpically disfavored in solvents like water
(Sen & Nielsen, 2006).
Although several thermodynamic models have been
described these models are mostly incomplete, very complex
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Fudan University on 05/07/15
and principally do not include the most important parameter
in FISH experiments, as FA. Therefore, more studies
including PNA components is needed in order to develop a
simple model capable of predicting complete thermodynamic
values of PNA-containing duplexes.
Software for thermodynamic prediction
There are different online programs which use thermo-
dynamic formulae for Tm calculation. The most usual
programs only give the user the Tm value, thus merely
allowing the selection of some parameters that influence this
temperature. Table 3 describes some of the programs that are
For personal use only.
available online, together with some of the characteristics
associated to each program.
The Oligonucleotide Properties Calculator software
(http://www.basic.northwestern.edu/biotools/oligocalc.html)
is a DNA and RNA specific software. In essence, this
program uses the strategy described in Breslauer et al. (1986)
to calculate thermodynamic properties and the values pub-
lished by Sugimoto et al. (1996).
The LNAÔ Oligo Tm Prediction tool (http://www.exiqon.-
com/ls/Pages/ExiqonTMPredictionTool.aspx) allow the pre-
diction of the duplex Tm between an oligonucleotide (LNA,
DNA or LNA:DNA) and a complementary DNA or RNA
strand.
The OligoAnalyzer software (http://eu.idtdna.com/analy-
zer/applications/oligoanalyzer/ default.aspx) estimates Tm
value from the NN model (SantaLucia et al., 1996) and
it uses the NN parameters already published for
DNA:DNA, RNA:DNA, RNA:RNA base pairs from Allawi
Table 3. Software for thermodynamic parameter prediction available online.
Software Adjustments
Oligonucleotide Properties Calculator software Salt and length
LNAÔ Oligo Tm Prediction – Only valid for neutral solutions (7–8), defined
OligoAnalyzer software Salt
mathFISH Salt, probe concentration, temperature
ProbeMelt Target molecule (RNA or microarrays)
Crit Rev Biotechnol, Early Online: 1–12
& SantaLucia (1997), Sugimoto et al. (1995), and Xia et al.
(1998), respectively. This is one of the most robust softwares
for Tm value prediction. There are other similar software
packages like PRIDE, PRIMO, AcePrimer and PrimeMaster,
however they generally produce deviations greater than, or
equal to 6.8  C (SantaLucia & Hicks, 2004).
An important tool in the FISH area was developed by
Yilmaz et al. (2011) and is a web-based program called
mathFISH (http://mathfish.cee.wisc.edu/) which incorporates
all previous models developed by the authors, specifically the
output model for the DGoverall and the linear energy model.
This program can simulate the hybridization between a given
target/probe sequence and gives the user functional param-
eters like the thermodynamic parameter set, hybridization
efficiency, affinity of a probe, and a predicted FA dissociation
profile of the probe. Furthermore, the program also informs
if the thermodynamic parameters of the probe are not the
most correct (Yilmaz et al., 2011). While the program does
not predict TH and provides no information on the properties
of PNA and LNA, it is certainly one of the most well adapted
for FISH procedures.
The most recent web tool was developed by Wright et al.
(2014) and it is called DECIPHER’s ProbeMelt (http://
decipher.cee.wisc.edu/ProbeMelt.html). With this software it
is possible to perform some relevant predictions in DNA
probes when FA is used. The mathematic modelling back-
ground used was based on the SRM model that has been
described above. The user is able to analyze hybridization
efficiencies using different levels of stringency and generating
denaturant curves. Additionally, ProbeMelt can be used in
combination with database searches, allowing the identifica-
tion of potential targets and nontargets (Wright et al., 2014).
Although it is a recent online tool, it is still only possible to
analyze DNA or RNA sequences and only the use of FA as a
denaturant is considered.
Melting temperature versus hybridization
temperature
In the previous chapter, we have addressed the thermo-
dynamic models more commonly used for theoretical predic-
tion of melting temperatures of DNA, PNA and LNA probes.
Despite the important information that we can obtain from
these models, we have shown above several reasons why the
Tm obtained from those calculations is still different from the
hybridization temperature of the probes. For a better
Notes
Only accepts symmetric sequences, fixed pH (pH
7.0), and denaturant agents are not included
oligonucleotides concentrations and specific salt
concentration.
The presence of denaturant agents is not included
Very complete software which performed all avail-
able simulations for a probe and a target/
nontarget sequence.
Predictions are performed for a defined temperature
(46  C) and specific salt concentration (1 M
sodium concentration).
 DOI: 10.3109/07388551.2014.993589
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Fudan University on 05/07/15
Figure 3. Predicted melting temperature (Tm) and respective hybridiza-
tion temperature (TH) at which the hybridization has been performed.
For more detailed information, see Tables S1, S2 and S3.
understanding of these differences we have identified studies
on FISH that used DNA, PNA and LNA probes and calculated
the melting temperature using the respective thermodynamic
models described above and compared it with the hybridiza-
tion temperature that was used by the authors (Figure 3).
More specifically, we used the parameters described by
Santalucia et al. (1996) and Equation (2) previously described
to determine the Tm for DNA probes. To calculate the Tm of
For personal use only.
PNA probes we used Equation (3) described above. The Tm
determination of LNA probes was performed based on
Owczarzy et al. (Owczarzy et al., 2011) parameters for
LNA using Equation (2). We did not use the models with
corrections for parameters such as salt concentration and
formamide. Relatively to the salt concentration, the majority
of the published results do not take these conditions in
consideration and their impact on the melting or hybridization
temperature is comparatively low. Regarding the formamide,
the correction models that exist were developed for DNA
probes, and since we address in this study not only DNA
probes but also other nucleic acid mimics probes the
application of the formamides models for these other probes
could originate misleading results.
In Figure 4, the average difference between the Tm
predicted for each type of probe, and the hybridization
temperature employed by the authors is represented. As
expected, analyses of Figures 3 and 4 show that in average the
TH is lower than the Tm for all types of probes. Another result
that can also be easily explained is the lower standard
deviation obtained for DNA and PNA probes, which is likely
to be due to the fact that PNA models are adaptations of the
DNA models and these DNA models and are much more
established than the recent developed models for LNA. LNA
probes show a higher variability than DNA and PNA probes,
with the TH being 7  C to 37  C lower that the predicted
melting temperature values. This variability may be due to the
fact that, unlike for DNA and PNA probes, many of the LNA
probes are constituted by DNA oligonucleotides with LNA
insertions. In adding to the number of LNA bases of the
probe, the positioning of the LNA bases within the probe also
has effects on the thermodynamics of the hybridization
(Kierzek et al., 2005), implying that Tm predictions are much
Melting temperatures in FISH 9
Figure 4. Difference between predicted melting temperature (Tm)
and hybridization temperature (TH). For more detailed information,
see Tables S1, S2 and S3.
harder to obtain for LNA probes. In addition, most of the
FISH methods performed using LNA are in animal cells. In
this case, many of the authors aim at developing the method
at 37  C, and as such might not be interested in identifying the
temperature at which the probe will work optimally.
The average difference between the TH and Tm of the PNA
probes was lower than the ones for DNA, which could be due
to the fact that no correction has been used for parameters
such as formamide. In our opinion, it is expected that different
types of nucleic acids have different (Tm-TH) values as the
slope of the melting temperature curve showed in Figure 1
might also vary for each nucleic acid. Nonetheless, because of
the lack of optimization and large scale verification of at least
some of the models, it is too early to draw any conclusions
in relation to the explicit differences between the various
probe chemistries.
Conclusions
FISH is a powerful molecular method used for the identifi-
cation and quantification of organisms of interest, both in
environmental and in medical samples. This technique has
evolved and become more powerful due to the appearance of
nucleic acid mimics that have substituted the traditional DNA
oligonucleotide probes used.
Despite this development, the optimization of this tech-
nique is still a trial and error process, in part due to the lack of
precise mathematic models to predict the hybridization
temperature of an oligonucleotide probe. The first thermo-
dynamic models were based on thermodynamic parameters
calculated for DNA or RNA oligonucleotides, but with the
appearance of the new DNA or RNA mimics it has become
necessary to adjust these models or to develop novel models.
The most used models to calculate thermodynamic param-
eters for DNA/RNA mimics oligonucleotides have been
derived from the ones used for DNA oligonucleotides
(Giesen et al., 1998; Kierzek et al., 2005; McTigue et al.,
2004) and provide important information about the conditions
at which the FISH procedure should be performed, helping to
decrease the time involved in method optimization. However,
 10 S. Fontenete et al.
the results often lack accuracy because these all models are
not specific for FISH since they were designed for other
molecular biology techniques, such as polymerase chain
reaction (PCR). Furthermore, they do not take into account all
parameters and chemical compounds involved in the hybrid-
ization step of a FISH procedure. If possible, one should
always confirm the predicted Tm values through two inde-
pendent models due to the occurrence of potential deviations
(Owczarzy et al., 1997). Furthermore, these methods only
allow us to predict the melting temperature which is used as a
reference for the hybridization temperature as illustrated
above when the Tm values predicted by the models were
compared with the TH’s used in the experiments.
FA and sodium ion concentrations are known to have an
impact on the hybridization reaction and therefore correction
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Fudan University on 05/07/15
models have been developed. Nevertheless, these models do
not give precise results for all kinds of oligonucleotide probes
and some of them involve complicated mathematical calcu-
lations and are therefore not practical for routine use for most
researchers. This obstacle may be alleviated by the use of
specialized software to predict the TH but at present such
software must be used carefully because most of them only
take a limited number of parameters of the hybridization
reaction into account.
In short, the thermodynamic models already developed for
DNA, RNA and DNA/RNA mimics only allow researchers to
obtain an indication of the range of the hybridization
For personal use only.
temperature, but a trial and error optimization will still be
required until the shortcomings identified in this review are
solved.
Declaration of interest
This work was funded by FEDER funds through the
Operational Programme for Competitiveness Factors –
COMPETE, ON.2 – O Novo Norte – North Portugal
Regional Operational Programme and National Funds through
FCT – Foundation for Science and Technology under the
projects: PEst-C/EQB/UI0511, NORTE-07-0124-FEDER-
000025 – RL2_ Environment&Health and DNA mimics
Research Project PIC/IC/82815/2007, PhD Fellowship SFRH/
BD/72999/2010 and Post-Doctoral fellowship SFRH/BPD/
78846/2011. The Nucleic Acid Center, University of Southern
Denmark is thanked for financial support.
JW is cofounder of RiboTask ApS which offer LNA/20 -
OMe-RNA probes for RNA targeting. NFA is cofounder of
Biomode SA which develops molecular methods for the rapid
detection of microorganisms.
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