The utility of formamide in the denaturation and renaturation of DNA has been examined. The melting temperature of duplex DNA is lowered by 0.6°C per per cent formamide. The depression of melting temperature is independent of the GC content. Formamide also increases the width of the thermal transition. Upto 30%, it does not affect the rate of DNA reassociation

Dutta, Santanu

Gopinathan, K. P.

Sadhu, Chanchal

English

Publications of the IAS Fellows

J. Biosci., Vol. 6, Number 6, December 1984, 817-821                               © Printed in India. 
 
 
 
 
 
 
Influence of formamide on the thermal stability of DNA 
 
 
 
CHANCHAL SADHU, SANTANU DUTTA* 
and K. P. GOPINATHAN† 
Microbiology and Cell Biology Laboratory, and *Molecular Biophysics Unit, Indian Institute 
of Science, Bangalore 560 012, India
 
MS received 5 May 1984; revised 20 August 1984 
 
Abstract. The utility of formamide in the denaturation and renaturation of DNA has been 
examined. The melting temperature of duplex DNA is lowered by 0·6oC per per cent 
formamide. The depression of melting temperature is independent of the GC content. 
Formamide also increases the width of the thermal transition. Upto 30%, it does not affect the 
rate of DNA reassociation. 
 
Keywords. DNA melting; formamide; Tm; DNA base composition; DNA reassociation. 
 
 
 
Introduction 
 
The optimum rate for DNA-DNA reassociation occurs at about 25°C below the 
melting temperature (Tm), (Wetmur and Davidson, 1968; Britten et al., 1974). The 
incubation temperature (Ti) for reassociation, on account of its dependence on Tm varies 
with the GC content of the DNA. For DNA samples of GC content of 50% or less. Ti is 
within the normally accessible range for optical and hydroxyapatite chromatographic 
studies. For instance, the optimum temperature for monitoring reassociation of 
Escherichia coli DNA (GC content, 50%) in the presence of 1Μ NaCl, is around 75°C. 
For DNA having 70% GC, in the presence of l M NaCl, Ti will be around 90°C. 
Incubation at such a high temperature often causes strand breakage and depurination 
of DNA. Hence the use of chemical agents whose presence lowers Tm without any 
specificity for base composition and does not hinder reassociation rates is advocated. 
 
Formamide seems to be the choicest of the agents and has been in use more than any 
other compound both in solution and in filter hybridization studies. The effect of 
formamide on the destabilization of DNA duplex was first studied systematically by 
McConaughy et al. (1969) using Bacillus subtilis DNA (40% GC). According to them 
the rate of reduction of Tm was 0·72°C per per cent formamide. Later, Casey and 
Davidson (1977) reported a value of 0·63°C for E. coli DNA. In this communication we 
report studies on the effect of formamide on denaturation of DNAs with a wider 
variation in base composition. 
 
 
† To whom reprint requests should be addressed. 
Abbreviations used: EDTA, Ethylenediamine-tetraacetic acid; RNA, ribonucleic acid; SDS, sodium dodecyl 
sulphate; Ti, temperature of incubation; Tm, temperature midpoint of melting. 
 
817 
818 Sadhu et al. 
 
Materials and methods 
 
Bacterial strains 
 
Mycobacterium smegmatis SN2, E. coli Β and Bacillus cereus were obtained from our 
laboratory culture collection. 
 
DNA isolation 
 
The cells were lysed by lysozyme-EDTA-sodium dodecyl sulphate (SDS), and the DNA 
was isolated by the phenol extraction procedure (Kirby, 1959). To achieve lysis of 
M. smegmatis cells sensitization of cells by pretreatment with glycine was necessary. 
Glycine (0·15 Μ) was added to the growing culture 2 h before harvesting. RNA was 
removed from the nucleic acid preparation by treatment with RNase A. In the case of 
B. cereus, a combination of RNase A and RNase T1 was used. After RNase digestion, 
sodium acetate was added (final concentration, 0·3 M) and the samples were extracted 
with (24:1) chloroform-isoamylakohol (Marmur, 1961). DNA was precipitated with 
2·5 volumes of ethanol, pooled and dissolved in 10mM Tris pH 7·0, containing 1 mM 
EDTA and 10mM NaCl.
 
Reagents 
 
Lysozyme, RNase A, RNase T1 formamide, Tris (hydroxymethyl) aminomethane and 
SDS were from Sigma Chemical Company, St. Louis, Missouri, USA. All other 
reagents were of analytical grade. Purification of formamide has been described 
elsewhere (Sadhu et al., 1984). 
 
Melting and reassociation of DNA 
 
Thermal denaturation was done according to the procedure described by Mandel and 
Marmur (1968) using a Pye-Unicam SP700A spectrophotometer fitted with an 
electrical heating system. Absorbance was monitored at 270 nm because of the inherent 
absorbance of formamide at 260 nm. For reassociation studies, stoppered cuvettes 
containing DNA was heated by circulating water. Reassociation was initiated by the 
addition of prewarmed sodium-phosphate buffer and the decrease in absorbance at 
270 nm was recorded for 1 h using Shimadzu ultra-violet recording spectrophotometer. 
In all the reassociation experiments Ti was maintained at 25°C below Tm at that 
condition. Results were plotted as suggested by Wetmur (1976). 
 
Results and discussion 
 
Melting of DNA in the presence of formamide 
 
The Tm values for M. smegmatis, Ε. coli and Β. cereus DNA in the absence of formamide 
were 90°C, 83·5°C and 78·5°C respectively (data not shown). Base compositions of these 
DNA species were determined from the equation (Mandel and Marmur, 1968): 
ΔGC = ΔTm × 2·44 using E. coli DNA as the standard. Assuming the GC content of E. 
coli DNA as 50% (Marmur, 1961) the GC contents of M. smegmatis and B. cereus DNA 
were found to be 66% and 38% respectively. 
Influence of formamide on the thermal stability of DNA                 819
 
Tm  values of each of these DNA samples in the presence of varying concentrations of
formamide were determined. With increasing concentration of formamide Tm values of
all the DNA samples were lowered. For each DNA sample, the Tm values were plotted
against formamide concentration (figure 1). The slopes of all the lines were same,
indicating that DNAs with varying GC content behave in an identical fashion in the 
presence of formamide. The drop in Tm with increasing concentration of formamide is 
linear and corresponds to 0·6°C per per cent formamide. As mentioned above, the 
DNAs employed in these studies varied in GC content from 38–66%. It is, therefore, 
possible that formamide does not interact with the bases in a differential manner. 
For each melting profile, the standard deviation, σ, which is a relative measure of 
cooperativity, was calculated by the equation (Wada et al., 1980): σ = (        -0.6)14ΔT .1
× 1·25. The results are presented in table 1. The value of σ increased with an increase in 
 
Figure 1. Effect of formamide on Tm of different DNA samples. (○), M. smegmatis; (●), E. 
coli; (Δ), Β. cereus. 
 
 
Table 1. Effect of formamide on σ value. 
 
(a), 0% formamide;   (b), 30% formamide; 
(c), 50% formamide; (d), 70% formamide. 
 
85.9
820 Sadhu et al. 
 
the formamide concentration. However, a quantitative correlation between GC 
content, σ values and formamide concentration could not be obtained. Cooperativity of 
the helix to coil transition in DNA is intensified by weakening the shielding effect of the 
ionic cloud (Wada et al., 1980). The shielding effect can be altered by changes in ionic 
strength, dielectric constant etc. At a fixed ionic strength, dielectric constant of the 
medium is increased from 81 of the aqueous phase to 96 by the addition of formamide 
(1:1, v/v) (Record, 1967). This may be the cause of the observed increase of σ in the 
presence of formamide. 
 
Effect of formamide on the rate of reassociation 
 
We have examined the effect of formamide on the rate of DNA reassociation using Ε. 
coli DNA. With increasing concentrations of formamide, reassociation was carried out 
at lower temperatures to maintain a difference of 25°C between Tm and Ti. The results 
are shown in figure 2. Upto 30%, formamide showed no appreciable effect on the rate of 
reassociation. At higher percentages of formamide, the reassociation rate was reduced. 
 
 
Figure 2. Effect of formamide on the rate of reassociation. Reassociation was carried out in 
the presence of 0·8 Μ phosphate buffer (pH 6.9). The Ti in the presence of 0, 15,30 and 45% of 
formamide were 78°C, 69°C, 60°C and 51°C respectively. ƒss, the fraction of single stranded 
DNA was calculated from the relation, l/ƒss = (Ao– Aα)/(At – Aα) were Ao is absorbance 
of entirely denatured molecules, Aα is absorbance at infinite time, and At is absorbance at 
time, t. (Δ), 0% formamide; (□), 15% formamide; (●), 30% formamide; (○), 45% formamide. 
 
Influence of formamide on the thermal stability of DNA                  821
 
The rate of reassociation in the presence of formamide can be influenced by the 
following factors: 
 
(i) Increased accessibility of the complementary strands to each other due to an 
increase in the dielectric constant of the medium causing a decrease in the interstrand 
electrostatic repulsion. 
(ii) Decreased bimolecular collisions due to the reduction of Ti. 
 
A balance between these two opposing factors probably kept the rate more or less 
unaltered upto 30% formamide. Further increase in formamide concentration may 
give rise to the condition where the temperature factor overtakes the former causing the 
observed decreased in the rate of reassociation. 
 
 
Acknowledgements 
 
We thank Dr. Hirak Basu for his keen interest in the work. Financial support from 
Department of Atomic Energy, Bombay and University Grants Commission, New 
Delhi, is gratefully acknowledged. 
 
 
References 
 
Britten, R. J., Graham, D. E. and Neufeld, Β. R. (1974) Methods Enzymol., 29, 363. 
Casey, J. and Davidson, N. (1977) Nucleic Acid. Res., 4, 1539. 
Kirby, K. S. (1959) Biochim. Biophys. Acta, 36, 117. 
Mandel, Μ. and Marmur, J. (1968) Methods Enzymol., 12B, 195. 
Marmur, J. (1961) J. Mol. Biol., 3, 208. 
McConaughy, Β. L, Laird, C D. and McCarthy, B. J. (1969) Biochemistry, 8, 3289. 
Record M. T. Jr. (1967) Biopolymer, 5, 975. 
Sadhu, C, Dutta, S. and Gopinathan, K. P. (1984) Anal. Biochcm. (In Press). 
Wada, Α., Yabuki, S. and Husimi, Y. (1980) CRC Crit. Rev. Biochem., 9, 87. 
Wetmur, J. G. (1976) Ann. Rev. Biophys. Bioeng., 5, 337. 
Wetmur, J. G. and Davidson, N. (1968) J. Mol. Biol., 31, 349. 


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