ANALYTICAL
BIOCHEMISTRY
94, 259-264 (1979)
Ethidium
Dimer: A New Reagent for the Fluorimetric Determination of Nucleic Acids
JUDITH
MARKOVITS," BERNARD P. ROQUES,?' JEAN-BERNARD LE PECQ*
AND
*Laboratoire de Physicochimie Macromokulaire LA (CNRS) 147, Institut Gustave-Roussy, 94800 Villejuif, France, and i‘Dkpartement de Chimie Organique, ERA (CNRS) 613 UER des Sciences Pharmaceutiques et Biologiques, 4 avenue de I’Observatoire, 75270 Paris Cedex 06, France Received August 2, 1978 A dimeric derivative of ethidium is used for fluorimetric assay of nucleic acids. Due to the very high binding affinity of this derivative for DNA and RNA, a significant increase of sensitivity of the ethidium fluorimetric technique for nucleic acids determination is obtained. Using a commercially available instrument, DNA concentrations in the nanogram per milliliter range are determined. In addition, we have found that an acridine ethidium dimer can also be used for a sensitive fluorimetric assay of DNA concentration, while simultaneously providing a determination of the DNA base composition.
A fluorimetric method for determining DNA and RNA concentrations using ethidium bromide was proposed by Le Pecq and Paoletti (1). The large fluorescence enhancement of ethidium on binding to double stranded nucleic acids has also been used to determine nucleic acids concentration in tissue homogenates (2,3). Several other dyes such as mithramycin (4,5) and 4’,6-diamidino-2-phenylindolee 2HCl (DAPI)’ (6) have similar properties and have also been proposed for the fluorimetric determination of DNA concentration. The sensitivity of this fluorimetric technique is mainly limited by the low binding affinity of these dyes for nucleic acids. Recently, it has been shown that bifunctional intercalating compounds have very high binding affinities for nucleic acids (7-9). It is shown here that if an ethidium homodimer is substituted for ethidium in the fluorimetric determination of nucleic acid concentrations, the sensitivity is greatly increased. ’ Abbreviation indole.2HCl.
used: DAPI,
diamidino-2-phenyl-
MATERIALS
AND METHODS
Chemicals. Structure of ethidium homodimer and acridine ethidium heterodimer are shown on Fig. 1. These compounds have been synthesized as already described (&lo). 4’,6-Diamidino-2-phenylindole.2HCl (DAPI) was purchased from Serva. Calf thymus, Clostridium perfringens , and Micrococcus luteus DNAs from Sigma were purified by three phenol extraction. Polyvinylsulfate, pancreatic neutral DNase (DNase
I), and
poly(A),
PO~YW),
poly(I),
poly(C), and bovine serum albumin fr. V were purchased from General Biochemicals, Worthington, and Miles respectively. Poly[d(A-T)]*poly[d(A-T)] was from Boehringer. Poly(A).poly(U) was obtained by mixing an equimolar solution of poly(A) and poly(U) at room temperature in 0.2 M Tris-HCl buffer, pH 7.4, containing 0.2 M NaCl. Formation of the duplex polymer was verified by the measurement of the hypochromicity at 260 nm. The preparations of rabbit blood plasma, hydrolyzed, and denatured DNAs were performed as described by Le Pecq et al. (1). 259
0003-2697/79/060259-06$02.00/O Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
260
MARKOVITS,
ROQUES,
AND
LE PECQ
2npLl”¶i3g”+4 Cl0 Ethidium
Acridi
FIG.
1. Chemical
ne
structure
homodimer
ethidium
of ethidium
heterodimer
homodimer
Fluorescence measurements. Fluorimetric measurements were made with a PerkinElmer MPF #A spectrofluorometer, fitted with a 150 W xenon lamp, and equipped with a sample chamber thermostatted at 25°C. DNA and dye concentrations were measured from their uv absorbance. General procedure. A freshly prepared stock solution of ethidium dimer in water (20 &ml) was diluted in the appropriate buffer and mixed with solutions of different DNA concentration. Fluorescence was measured after 5 min equilibration. Standard conditions are: fluorescence exitation at 370 nm using a 16-nm slit, and fluorescence emission at 620 nm with a 16-nm slit. Second-order spectrum is eliminated by interposition of the built-in 43 filter. RESULTS
As shown previously (1) in presence of saturating concentrations of ethidi?lm bromide the fluorescence intensity increases linearly with the nucleic acid concentration.
and acridine
ethidium
heterodimer.
For a dye which binds to nucleic acids with concomitant variations in the dye fluorescence the following equations apply (11). 11 - 10 = k(Vh - l)Cb, IO = kCt,
(1) (2)
where I1 and I0 are the fluorescence intensities of a given solution of dye at concentration Ct, respectively in presence and in absence of nucleic acids. VA is the ratio: fluorescence intensity emitted by the bound dye over fluorescence intensity emitted by the free dye, when fluorescence measurements are made under identical conditions at a given excitation wavelength, h. Cb is the concentration of bound dye and k is a constant depending both on instrumental sensitivity and on the spectroscopic characteristics of the dye. If the dye concentration is in large excess such that nucleic acids are fully saturated: Cb=CNA xn, (3) where CNA is the nucleic acid concentration,
NUCLEIC
ACIDS
FLUORESCENCE
and n is the number of binding sites for the dye to the type of nucleic acid under study. From equations 1, 2 and 3 it follows that I, - IO = k(Vh - 1) x n x C&4.
(4)
The relative increase of fluorescence per unit of nucleic acid concentration measures the sensitivity of the method:
-c (“,‘I/
(V, - 1) x n
0 NA
=
(5)
The fluorimetric determination of nucleic acids according to this principle will therefore be the most sensitive, when Vh is large and when the concentration of dye used is as small as possible. But Ct cannot be smaller than a certain value let us say 10 x K-l, (K-l being the dissociation constant of the dye-nucleic acid complex) otherwise the nucleic acid is not saturated by the dye (1) and Eq. (3) no longer applies. For a dye of infinitely small dissociation constant the method is then obviously limited by the necessity of measuring Z, and IO values significantly different from the background fluorescence. As shown previously (1): (6)
where qb and qf are the quantum yield Of fluorescence of the bound and free dye respectively and Eb and Ef the respective absorption coefficients in the bound and free state. Equation 2 can be written Zo = kinst X Ef X qf,
(7)
where kinst is a constant depending only on the instrument performance. Combining Eqs. (4), (6), and (7) and assuming VA * 1: ZI-Zo=ki,,tXEbXqbXnXCNA.
(8)
It can therefore be concluded that for a dye of limited binding affinity for nucleic acids, the sensitivity of the method is limited
ASSAY
261
by the necessity to use high concentration of dye [Eq. (5)]. For a dye of very high binding affinity the method is only limited by the instrument sensitivity and by the intensity of the background fluorescence. Therefore the ideal dye for the fluorimetric determination of nucleic acids will be a dye having a very high binding affinity for nucleic acids, high quantum yield, and extinction coefficient in the bound state, with low quantum yield and extinction coefficient in the free state. When ethidium bromide is used, the sensitivity is limited by the relatively low value of the dissociation constant of the nucleic acid-dye complex. As already discussed (12), nucleic acids cannot readily be determined below concentrations of 0.1 pg/ml with this dye. Recently we have prepared bifunctional intercalators and we have shown that such molecules have binding affinities for nucleic acids which are 1000 to 10,000 times higher than the monomeric derivatives. In particular, a dimeric derivative of ethidium (Fig. 1) has been synthesized and studied (8-10). This molecule has a binding affinity much larger than ethidium bromide for DNA and RNA. This molecule could therefore represent a better dye for fluorimetric determination of nucleic acids. To determine the best conditions for the use of ethidium dimer, the variations of the value of Vh as a function of the wavelength of excitation were first measured (Fig. 2). The results show that two wavelengths are to be preferred (370 and 550 nm) for fluorescence excitation. We selected in this work 370 nm for the excitation wavelength because of the larger extinction coefficient of the dye at this wavelength and for better sensitivity of our instrument under these conditions. On the other hand it is observed that the concentration of dye to be used can be lowered as expected without changing the slope of the curve I, - IO versus CNA (Fig. 3) [Eq. (5)]. Using buffer concentrations cor-
MARKOVITS,
262
300
1
ROQUES, AND LE PECQ
I
I
I
400 Wavalm#hlnml
I
500
FIG. 2. Variation of VAas a function of excitation wavelength, (A,, = 620 nm). The Perkin-Elmer instrument was used in the ratio mode. In the monitor chamber cover we replaced the diffusion plate by a reference cell containing 1 &ml of ethidium dimer The sample cell contained 0.5 pg/ml of native calf thymus DNA and 150 @ml of ethidium dimer in 0.05 1w Tris-HCl buffer, 0.05 M NaCI, pH 7.4. VA was independently determined at 365, 405, 436, and 546 nm and the values obtained were used for calibration.
responding to physiological conditions, the sensitivity of the determination using this dye is then only limited by the instrument sensitivity and the background fluorescence of the buffer. It is apparent that a DNA concentration as low as 1 rig/ml can then be determined, a value 100 times smaller than the concentration which can be determined using ethidium bromide. It has been verified (Table 1) that the specificity of interaction of ethidium bromide for double stranded nucleic acids is retained for this ethidium dimer. In particular hydrolyzed DNA and RNA do not interfere such that the procedure described (12) for the determination of DNA and RNA in a mixture can be used, even in a complex medium. We have recently described an acridine ethidium heterodimer (Fig. 1). This molecule has also a high affinity for nucleic acids. If its fluorescence is excited in the phenan-
thridinium band (550 nm) it can be used in the same manner and with about the same sensitivity as the ethidium dimer as described here. It was also shown (9) that the ratio of fluorescence excited at 550 nm over the fluorescence excited at 470 nm in the acridine absorption range permits the estimation of DNA base composition. With this molecule, DNA concentration and DNA base composition can be estimated on quantities smaller than 10 ng. DISCUSSION
It is shown that by substituting an ethidium dimer for ethidium bromide, the sensitivity of the fluorimetric determination of nucleic acids can be increased lOO-fold and that DNA concentrations as low as 1 &ml can be estimated. Kapuscinski et al. (6) recently proposed the 4’ ,6-diamidino-2-phenylindole * 2HCl (DAPI) for the fluorimetric measurement of DNA concentration. Although DNA con-
NUCLEIC
I 0
ACIDS FLUORESCENCE
I
ASSAY
263
I
5
10 DNA
concentrat~on(nglml)
FIG. 3. Effect of ethidium dimer concentration on the sensitivity of the fluorescence assay of DNA. Fluorescence intensity of solutions (corrected from background fluorescence) is measured as a function of DNA concentrations, using different concentrations of ethidium dimer (50, 100, 200, and 400 rig/ml EtDi) as shown on the figure. Solutions were in 0.05 M Tris-HCl buffer, pH 7.4, 0.05 M NaCl. The fluorescence intensity of the solutions containing 50 &ml of ethidium dimer was only 30% higher than background fluorescence. Lower concentrations have therefore not been used.
centrations close to 1 rig/ml could be determined using DAPI, this compound is only fluorescent in the presence of DNA and not in the presence of RNA. The use of DAPI is therefore limited to the determination of DNA concentration. However, it is also important to note that the DNA binding and fluorescence properties of DAPI are very sensitive to DNA base composition. For instance, we have observed that no fluorescence increase of DAPI is obtained in presence of DNA of high Gua + Cyt content, (Mic~ococc~~ luteus DNA; results not shown). As previously observed (6) the binding heterogeneity of DAPI to DNA regions of different Gua + Cyt content is accompanied by changes in the fluorescence properties of the dye. This explains why
the variation of fluorescence of this dye with DNA concentration is not perfectly linear. Therefore, the use of this dye for DNA fluorimetric determination is limited to DNA of high Ade + Thy content. Furthermore, measurements of DNA concentrations can only be performed using DAPI if the DNA base composition is known. On the contrary, the fluorescence properties of the DNA-bound ethidium dimer are not sensitive to the base composition of DNA (similar to ethidium bromide fluorescence). However, as for ethidium (12), the fluorescence of ethidium dimer is a little higher when it is bound to poly[d(A-T)] * poly[d(A-T)]. This phenomenon is probably related to the special structure of this copolymer. Calibration curves can therefore be made with
MARKOVITS,
264 TABLE
ROQUES, AND LE PECQ
1
FLUORESCENT INCREMENT (Z1 - 4) OF ETHIDIUM DIMER IN THE PRESENCE OF A VARIETY OF SUBSTANCES~
Products
f, - 10
Calf thymus DNA Clostridium Micrococcus
perfringens luteus DNA
100
DNA
100 loo
Poly[d(A-T)] .poly[d(A-T)] Denatured DNA DNase hydrolyzed DNA
105 47 0.1
Pol~W.polyW)
190
POlY(I) Poly(A)
180 1.7
POlY(C)
POlYW)
ACKNOWLEDGMENTS The authors are very grateful to R. Oberlin and B. Gaugain for providing the samples of ethidium homodimer and acridine ethidium heterodimer. We thank Dr. J. Barbet for helpful discussions and C. G. Reinhardt for critical evaluation of the manuscript.
REFERENCES 1.
1.1
4.5 0.5 co.005
BIOCHEMISTRY
94, 259-264 (1979)
Ethidium
Dimer: A New Reagent for the Fluorimetric Determination of Nucleic Acids
JUDITH
MARKOVITS," BERNARD P. ROQUES,?' JEAN-BERNARD LE PECQ*
AND
*Laboratoire de Physicochimie Macromokulaire LA (CNRS) 147, Institut Gustave-Roussy, 94800 Villejuif, France, and i‘Dkpartement de Chimie Organique, ERA (CNRS) 613 UER des Sciences Pharmaceutiques et Biologiques, 4 avenue de I’Observatoire, 75270 Paris Cedex 06, France Received August 2, 1978 A dimeric derivative of ethidium is used for fluorimetric assay of nucleic acids. Due to the very high binding affinity of this derivative for DNA and RNA, a significant increase of sensitivity of the ethidium fluorimetric technique for nucleic acids determination is obtained. Using a commercially available instrument, DNA concentrations in the nanogram per milliliter range are determined. In addition, we have found that an acridine ethidium dimer can also be used for a sensitive fluorimetric assay of DNA concentration, while simultaneously providing a determination of the DNA base composition.
A fluorimetric method for determining DNA and RNA concentrations using ethidium bromide was proposed by Le Pecq and Paoletti (1). The large fluorescence enhancement of ethidium on binding to double stranded nucleic acids has also been used to determine nucleic acids concentration in tissue homogenates (2,3). Several other dyes such as mithramycin (4,5) and 4’,6-diamidino-2-phenylindolee 2HCl (DAPI)’ (6) have similar properties and have also been proposed for the fluorimetric determination of DNA concentration. The sensitivity of this fluorimetric technique is mainly limited by the low binding affinity of these dyes for nucleic acids. Recently, it has been shown that bifunctional intercalating compounds have very high binding affinities for nucleic acids (7-9). It is shown here that if an ethidium homodimer is substituted for ethidium in the fluorimetric determination of nucleic acid concentrations, the sensitivity is greatly increased. ’ Abbreviation indole.2HCl.
used: DAPI,
diamidino-2-phenyl-
MATERIALS
AND METHODS
Chemicals. Structure of ethidium homodimer and acridine ethidium heterodimer are shown on Fig. 1. These compounds have been synthesized as already described (&lo). 4’,6-Diamidino-2-phenylindole.2HCl (DAPI) was purchased from Serva. Calf thymus, Clostridium perfringens , and Micrococcus luteus DNAs from Sigma were purified by three phenol extraction. Polyvinylsulfate, pancreatic neutral DNase (DNase
I), and
poly(A),
PO~YW),
poly(I),
poly(C), and bovine serum albumin fr. V were purchased from General Biochemicals, Worthington, and Miles respectively. Poly[d(A-T)]*poly[d(A-T)] was from Boehringer. Poly(A).poly(U) was obtained by mixing an equimolar solution of poly(A) and poly(U) at room temperature in 0.2 M Tris-HCl buffer, pH 7.4, containing 0.2 M NaCl. Formation of the duplex polymer was verified by the measurement of the hypochromicity at 260 nm. The preparations of rabbit blood plasma, hydrolyzed, and denatured DNAs were performed as described by Le Pecq et al. (1). 259
0003-2697/79/060259-06$02.00/O Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
260
MARKOVITS,
ROQUES,
AND
LE PECQ
2npLl”¶i3g”+4 Cl0 Ethidium
Acridi
FIG.
1. Chemical
ne
structure
homodimer
ethidium
of ethidium
heterodimer
homodimer
Fluorescence measurements. Fluorimetric measurements were made with a PerkinElmer MPF #A spectrofluorometer, fitted with a 150 W xenon lamp, and equipped with a sample chamber thermostatted at 25°C. DNA and dye concentrations were measured from their uv absorbance. General procedure. A freshly prepared stock solution of ethidium dimer in water (20 &ml) was diluted in the appropriate buffer and mixed with solutions of different DNA concentration. Fluorescence was measured after 5 min equilibration. Standard conditions are: fluorescence exitation at 370 nm using a 16-nm slit, and fluorescence emission at 620 nm with a 16-nm slit. Second-order spectrum is eliminated by interposition of the built-in 43 filter. RESULTS
As shown previously (1) in presence of saturating concentrations of ethidi?lm bromide the fluorescence intensity increases linearly with the nucleic acid concentration.
and acridine
ethidium
heterodimer.
For a dye which binds to nucleic acids with concomitant variations in the dye fluorescence the following equations apply (11). 11 - 10 = k(Vh - l)Cb, IO = kCt,
(1) (2)
where I1 and I0 are the fluorescence intensities of a given solution of dye at concentration Ct, respectively in presence and in absence of nucleic acids. VA is the ratio: fluorescence intensity emitted by the bound dye over fluorescence intensity emitted by the free dye, when fluorescence measurements are made under identical conditions at a given excitation wavelength, h. Cb is the concentration of bound dye and k is a constant depending both on instrumental sensitivity and on the spectroscopic characteristics of the dye. If the dye concentration is in large excess such that nucleic acids are fully saturated: Cb=CNA xn, (3) where CNA is the nucleic acid concentration,
NUCLEIC
ACIDS
FLUORESCENCE
and n is the number of binding sites for the dye to the type of nucleic acid under study. From equations 1, 2 and 3 it follows that I, - IO = k(Vh - 1) x n x C&4.
(4)
The relative increase of fluorescence per unit of nucleic acid concentration measures the sensitivity of the method:
-c (“,‘I/
(V, - 1) x n
0 NA
=
(5)
The fluorimetric determination of nucleic acids according to this principle will therefore be the most sensitive, when Vh is large and when the concentration of dye used is as small as possible. But Ct cannot be smaller than a certain value let us say 10 x K-l, (K-l being the dissociation constant of the dye-nucleic acid complex) otherwise the nucleic acid is not saturated by the dye (1) and Eq. (3) no longer applies. For a dye of infinitely small dissociation constant the method is then obviously limited by the necessity of measuring Z, and IO values significantly different from the background fluorescence. As shown previously (1): (6)
where qb and qf are the quantum yield Of fluorescence of the bound and free dye respectively and Eb and Ef the respective absorption coefficients in the bound and free state. Equation 2 can be written Zo = kinst X Ef X qf,
(7)
where kinst is a constant depending only on the instrument performance. Combining Eqs. (4), (6), and (7) and assuming VA * 1: ZI-Zo=ki,,tXEbXqbXnXCNA.
(8)
It can therefore be concluded that for a dye of limited binding affinity for nucleic acids, the sensitivity of the method is limited
ASSAY
261
by the necessity to use high concentration of dye [Eq. (5)]. For a dye of very high binding affinity the method is only limited by the instrument sensitivity and by the intensity of the background fluorescence. Therefore the ideal dye for the fluorimetric determination of nucleic acids will be a dye having a very high binding affinity for nucleic acids, high quantum yield, and extinction coefficient in the bound state, with low quantum yield and extinction coefficient in the free state. When ethidium bromide is used, the sensitivity is limited by the relatively low value of the dissociation constant of the nucleic acid-dye complex. As already discussed (12), nucleic acids cannot readily be determined below concentrations of 0.1 pg/ml with this dye. Recently we have prepared bifunctional intercalators and we have shown that such molecules have binding affinities for nucleic acids which are 1000 to 10,000 times higher than the monomeric derivatives. In particular, a dimeric derivative of ethidium (Fig. 1) has been synthesized and studied (8-10). This molecule has a binding affinity much larger than ethidium bromide for DNA and RNA. This molecule could therefore represent a better dye for fluorimetric determination of nucleic acids. To determine the best conditions for the use of ethidium dimer, the variations of the value of Vh as a function of the wavelength of excitation were first measured (Fig. 2). The results show that two wavelengths are to be preferred (370 and 550 nm) for fluorescence excitation. We selected in this work 370 nm for the excitation wavelength because of the larger extinction coefficient of the dye at this wavelength and for better sensitivity of our instrument under these conditions. On the other hand it is observed that the concentration of dye to be used can be lowered as expected without changing the slope of the curve I, - IO versus CNA (Fig. 3) [Eq. (5)]. Using buffer concentrations cor-
MARKOVITS,
262
300
1
ROQUES, AND LE PECQ
I
I
I
400 Wavalm#hlnml
I
500
FIG. 2. Variation of VAas a function of excitation wavelength, (A,, = 620 nm). The Perkin-Elmer instrument was used in the ratio mode. In the monitor chamber cover we replaced the diffusion plate by a reference cell containing 1 &ml of ethidium dimer The sample cell contained 0.5 pg/ml of native calf thymus DNA and 150 @ml of ethidium dimer in 0.05 1w Tris-HCl buffer, 0.05 M NaCI, pH 7.4. VA was independently determined at 365, 405, 436, and 546 nm and the values obtained were used for calibration.
responding to physiological conditions, the sensitivity of the determination using this dye is then only limited by the instrument sensitivity and the background fluorescence of the buffer. It is apparent that a DNA concentration as low as 1 rig/ml can then be determined, a value 100 times smaller than the concentration which can be determined using ethidium bromide. It has been verified (Table 1) that the specificity of interaction of ethidium bromide for double stranded nucleic acids is retained for this ethidium dimer. In particular hydrolyzed DNA and RNA do not interfere such that the procedure described (12) for the determination of DNA and RNA in a mixture can be used, even in a complex medium. We have recently described an acridine ethidium heterodimer (Fig. 1). This molecule has also a high affinity for nucleic acids. If its fluorescence is excited in the phenan-
thridinium band (550 nm) it can be used in the same manner and with about the same sensitivity as the ethidium dimer as described here. It was also shown (9) that the ratio of fluorescence excited at 550 nm over the fluorescence excited at 470 nm in the acridine absorption range permits the estimation of DNA base composition. With this molecule, DNA concentration and DNA base composition can be estimated on quantities smaller than 10 ng. DISCUSSION
It is shown that by substituting an ethidium dimer for ethidium bromide, the sensitivity of the fluorimetric determination of nucleic acids can be increased lOO-fold and that DNA concentrations as low as 1 &ml can be estimated. Kapuscinski et al. (6) recently proposed the 4’ ,6-diamidino-2-phenylindole * 2HCl (DAPI) for the fluorimetric measurement of DNA concentration. Although DNA con-
NUCLEIC
I 0
ACIDS FLUORESCENCE
I
ASSAY
263
I
5
10 DNA
concentrat~on(nglml)
FIG. 3. Effect of ethidium dimer concentration on the sensitivity of the fluorescence assay of DNA. Fluorescence intensity of solutions (corrected from background fluorescence) is measured as a function of DNA concentrations, using different concentrations of ethidium dimer (50, 100, 200, and 400 rig/ml EtDi) as shown on the figure. Solutions were in 0.05 M Tris-HCl buffer, pH 7.4, 0.05 M NaCl. The fluorescence intensity of the solutions containing 50 &ml of ethidium dimer was only 30% higher than background fluorescence. Lower concentrations have therefore not been used.
centrations close to 1 rig/ml could be determined using DAPI, this compound is only fluorescent in the presence of DNA and not in the presence of RNA. The use of DAPI is therefore limited to the determination of DNA concentration. However, it is also important to note that the DNA binding and fluorescence properties of DAPI are very sensitive to DNA base composition. For instance, we have observed that no fluorescence increase of DAPI is obtained in presence of DNA of high Gua + Cyt content, (Mic~ococc~~ luteus DNA; results not shown). As previously observed (6) the binding heterogeneity of DAPI to DNA regions of different Gua + Cyt content is accompanied by changes in the fluorescence properties of the dye. This explains why
the variation of fluorescence of this dye with DNA concentration is not perfectly linear. Therefore, the use of this dye for DNA fluorimetric determination is limited to DNA of high Ade + Thy content. Furthermore, measurements of DNA concentrations can only be performed using DAPI if the DNA base composition is known. On the contrary, the fluorescence properties of the DNA-bound ethidium dimer are not sensitive to the base composition of DNA (similar to ethidium bromide fluorescence). However, as for ethidium (12), the fluorescence of ethidium dimer is a little higher when it is bound to poly[d(A-T)] * poly[d(A-T)]. This phenomenon is probably related to the special structure of this copolymer. Calibration curves can therefore be made with
MARKOVITS,
264 TABLE
ROQUES, AND LE PECQ
1
FLUORESCENT INCREMENT (Z1 - 4) OF ETHIDIUM DIMER IN THE PRESENCE OF A VARIETY OF SUBSTANCES~
Products
f, - 10
Calf thymus DNA Clostridium Micrococcus
perfringens luteus DNA
100
DNA
100 loo
Poly[d(A-T)] .poly[d(A-T)] Denatured DNA DNase hydrolyzed DNA
105 47 0.1
Pol~W.polyW)
190
POlY(I) Poly(A)
180 1.7
POlY(C)
POlYW)
ACKNOWLEDGMENTS The authors are very grateful to R. Oberlin and B. Gaugain for providing the samples of ethidium homodimer and acridine ethidium heterodimer. We thank Dr. J. Barbet for helpful discussions and C. G. Reinhardt for critical evaluation of the manuscript.
REFERENCES 1.
1.1
4.5 0.5 co.005
