ANALYTICAL
83, 252-257(1977)
BIOCHEMISTRY
Simple
and Rapid Fluorimetric DNA Microassay
JAN KAPU&X~SKI* *Technical
University,
Radom,
AND BOGNA
Method
for
SKOCZYLAS~'
and tM. Nencki Institute of Experimental Str., 02-093 Warsaw, Poland
Biology,
3 Pasteur
Received December 6, 1976; accepted June 20, 1977 DEDICATEDTOPROFESSORWLODZIMIERZ
NIEMIERKOON
HIS~OTH
ANNIVERSARY
4’,6-Diamidino-2-phenylindole.2HCl (DAPI) forms a specific complex with DNA, and this fact has been used for the quantitative fluorimetric estimation of DNA. The method permits the estimation of concentrations of DNA as low as 5 x IO-lo g/ml, even in the presence of a 20-fold excess of RNA. Complex formation depends on the DNA to DAPI ratio, DNA structure, ionic strength, and the presence of essential bivalent anions and cations.
In 1975, Russel et al. (1) and Williamson and Fennel (2) showed that 4’,6-diamidino-2-phenylindole.2HCl (DAPI) reacts with DNA to form a fluorescent complex. They proposed that this compound could make DNA visible in cells infected with mycoplasma and/or viruses. There are bases for assumption that DAPI exhibits a preference for several continuous A-T base pairs (3-5). After experimental confirmation of the above phenomenon (6,7) we decided to investigate the mechanism of the DAPIpolynucleotides interaction. The specific intercalation of DAPI to DNA, which does not occur between DAPI and RNA (8,9), has enabled us to develop a simple fluorimetric micromethod for DNA analysis. The method appeared to be much more sensitive than those previously published. The lowest DNA concentration which can be detected as a DAPI-DNA complex, under the conditions given below, is 5 X lo-lo g/ml. Other fluorescent complexes have been used previously (lo- 12); however, only one of the methods, that described by Hill and Whatley (10) employing the antibiotic Mithramycin, is really simple and specific for DNA, but its sensitivity is limited to 5 x 10e7 g of DNA/ml. MATERIALS
AND METHODS
Equipment. The excitation and fluorescence spectra were obtained using an MPF-3 Hitachi-Perkin-Elmer spectrophotometer equipped with an R106 photomultiplier tube and a 150-W xenon lamp. Measurements of ’ To whom correspondence
should be addressed. 252
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 0003.2697
SIMPLE
AND
RAPID
DNA
MICROASSAY
253
DNA-DAPI complex were made at wavelengths of 372 and 454 nm, representing maxima of excitation and fluorescence, respectively. The fluorescence intensity of the examined sample, i,, was compared with is, the fluorescence intensity of Hitashi-Perkin-Elmer standard No. 4 in order to obtain reproducible results, independent of the width of the entrance and exit slits. The measurements of standard No. 4 were made at an excitation wavelength of 365 nm with equally wide slits. The relative fluorescence intensity has been expressed as i = (i,li& x 104. The sum of the relative fluorescence intensities of all components has been compared with i, (blank test). Measurements were made in l-cm--path silica cells using filter 39 to eliminate light dispersion. Reagents. DAPI was synthesized according to the procedure of Dann et al. (13) with modification (6); uv maxima = 224, 261, and 344 nm, and r = 23,500, 18,600, and 23,000, respectively. DAPI was dissolved in water to a concentration of 5.71 PM (2 pg/ml). This solution had an excitation maximum at wavelength 358 nm and a relative fluorescence intensity of i = 213.2 (when measured at the fluorescence maximum of 449 nm). Presently DAPI can be supplied by Serva Feinbiochemica, D.69 Heidelberg 1, P.O. Box 105260. Preparation of polynucleotides. The stock solution of DNA at a concentration of 250 pg/rnl was dissolved in 0.01 M NaCl (unless stated otherwise in the text). Dilutions of this DNA stock solution were also made with 0.01 M NaCl. DNA denaturation was carried out using a fivefold diluted DNA solution buffered with 0.005 M Hepes, pH 7. The samples were heated for 10 min at 100°C and were immediately chilled in ice. The degradation of stock solution to yield a sheared DNA preparation was carried out by using either (i) an MSE ultrasonicator (30-set sonications were repeated four times at 30-set intervals) or (ii) expelling the DNA solution through syringe needle No. 27 (14). Chemicals. All chemicals used were of high commercial purity and were tested for the presence of fluorescent impurities. The relative fluorescence intensity of 0.015 M solutions was between 1.0 and 5.0. General procedure. The solution of DAPI in water was mixed with the DNA solution in 0.01 M NaCl or 0.0066 M Na,SO, and was buffered with Tris or Hepes, at a final concentration of 0.005 M, to pH 7. These samples were then ready for measurement immediately after mixing or could be stored refrigerated for several weeks without any change in their optical properties prior to estimation. RESULTS
AND DISCUSSION
Figure 1 presents a comparison of the excitation and fluorescence spectra of the mixture of DNA and DAPI with the spectra of a DAPI solu-
254
KAPUSCIfiSKI
AND SKOCZYLAS
15
?O(
300
400
500
20
2.5
30
600 ml
FIG. 1. The excitation spectrum: (la) DNA-DAPI complex, (lb) DAPI solution. The fluorescence spectrum: (2a) DNA-DAPI complex, (2b) DAPI solution. DNA concentration in samples la and 2a is 20 ).&ml. DAPI concentration in all samples is 2 &ml. Samples are in 0.012 M NaCl, 0.05 M Hepes, pH 7. The excitation spectrum measurements (the excitation slit: 1 nm) were made at a fluorescence wavelength of 454 nm (emission slit: 20 nm). The fluorescence spectrum measurements (emission slit: 2 nm) were made at an excitation wavelength of 372 nm (excitation slit: 6 nm). Recorder sensitivity: 10; scan speed: 150 nm/min; chart speed: 40 mm/min. Inset: The influence of [DNA]/[DAPI] = I< on W (fluorescence intensification of the complex). [DNA] = 143 PM (50 &ml). Samples are in 0.012 M NaCl, 0.005 M Hepes, pH 7.
tion. There is a distinct fluorescence intensification of the mixture compared to the fluorescence of the individual components (the DNA solution showed no significant fluorescence under these experimental conditions). The fluorescence intensification, W = (i - i,)I[DAPI], depends on the ratio of components, R = [DNA]/[DAPI]. The strongest fluorescence intensification was obtained when R > 100 nucleotides/l molecule of DAPI, which is shown in the inset to Fig. 1. Practically the whole quantity of DAPI forms the fluorescent complex with DNA in this range of concentrations. The value of fluorescent intensification ( W) is also a function of the absolute DNA concentration. The highest values of W are reached by increasing values of R, if the DNA concentration
SIMPLE AND RAPID DNA MICROASSAY
255
increases. In very diluted samples, however, the highest value of W is reached at R > 5. Therefore, perhaps unexpectedly, the more diluted the DNA and DAPI solutions, the easier the formation of the fluorescent complex. For analytical purposes, a range of [DNA]/[DAPI] ratios should be used within which the fluorescent intensification (W) does not reach the maximal value. Therefore, the range of R = 0.28-5.5 for DNA concentrations of 5 x lo-lo to 1 x 10-j g/ml has been used with four DAPI concentrations (Fig. 2a-d). These results show that this method is useful for quantitative DNA assays over a wide range of DNA concentrations above 0.5 &ml. The ranges of DNA concentrations used in Fig. 2b and c, namely 5-1000 @ml, seem to be the most advantageous, because of their almost linear dependence. Various conditions can influence the fluorescence intensity in this method, and these are discussed below. Salt Concentration Increasing the ionic strength of the solutions reduces the fluorescence of the DAPI-DNA complex (Na+, chloride, perchlorate, acetate, EDTA). For accuracy, the ionic strength of the solutions used should be below 0.1. The ions of bivalent metals such as 2 x 10e3 M Mg2+, 1 x 10e3 M
FIG. 2. The calibration curves for quantitative DNA assay in complex with DAPI at varying DAPI concentrations applied to four different ranges of DNA concentration. i is the relative fluorescence intensity (i = (iA&) x 104; see Results and Discussion). DNA concentration in rig/ml = 0.5-10, 5-100, 50-1000, and 500-10,000 in a-d, respectively. DAPI concentration in @ml = 2, 20, 200, and 2000 in a-d, respectively. The samples are in 0.012M NaCl, 0.005 M Hepes, pH 7.
256
KAPUSCINSKI
AND SKOCZYLAS
Mn2+, 1 x 1O-3 M Ca2+ and anions such as 1 x 10-l M citrates and 1 x 10d3 M HPOa2- reduce fluorescence by a factor of approximately 2 in relation to NaCl solution of the same ionic strength. However, the fluorescence increases significantly, by a factor of approximately 2, when SOa2- ions are substituted for Cl- ions. The most suitable buffers for the assay were Tris, Hepes, and EDTA. Dependence
of Fluorescence
Intensity
on DNA Structure
The fluorescence intensity is highest when DAPI is complexed with native highly polymerized DNA and decreases when DAPI is complexed with degraded or denaturated DNA (see Table 1). However, this method may be used to analyze all these forms of DNA, if individual calibration curves are made. TABLE COMPARISON
1
OF THE RELATIVE FLUORESCENCE INTENSITIES OF DIFFERENT THE WHOLE HISTONE FRACTION, NUCLEOTIDES, AND UREA COMPLEXED WITH DAPI”
KINDS
OF
DNA AND RNA,
i - i.
Materials
Calf thymus high-polymerized, type I” Native DNA Native DNA sheared Native DNA sonicated Denaturated DNA Calf thymus high-polymerized native DNA” Herring high-polymerized native DNAd Calf thymus degraded DNAe Herring degraded DNA, type IV* Yeast high-polymerized RNA, type XI* Yeast high-polymerized RNA’ Yeast degraded RNAe Calf thymus high-polymerized native DNA, type Ib +RNA (type XI),* 1:l (w/w) +RNA (type XI),* 1:20 (w/w) +Histone,’ 1:l (w/w) +Urea, 0.1 M Nucleotides: AMP,’ GMP,’ UMP,d CMP” (each) Deoxynucleotides: * d-AMP, d-GMP, d-CMP, d-TMP (each) a The relative fluorescence complex is regarded as 100%. b Sigma Chemical Co. c Serva. d Calbiochem. e BDH. f PGCH. 0 FLUKA. h REANAL.
intensity
of the high-polymerized
100 94 74 33 100 102 38 9 3 1 3 97 92 92 116
83, 252-257(1977)
BIOCHEMISTRY
Simple
and Rapid Fluorimetric DNA Microassay
JAN KAPU&X~SKI* *Technical
University,
Radom,
AND BOGNA
Method
for
SKOCZYLAS~'
and tM. Nencki Institute of Experimental Str., 02-093 Warsaw, Poland
Biology,
3 Pasteur
Received December 6, 1976; accepted June 20, 1977 DEDICATEDTOPROFESSORWLODZIMIERZ
NIEMIERKOON
HIS~OTH
ANNIVERSARY
4’,6-Diamidino-2-phenylindole.2HCl (DAPI) forms a specific complex with DNA, and this fact has been used for the quantitative fluorimetric estimation of DNA. The method permits the estimation of concentrations of DNA as low as 5 x IO-lo g/ml, even in the presence of a 20-fold excess of RNA. Complex formation depends on the DNA to DAPI ratio, DNA structure, ionic strength, and the presence of essential bivalent anions and cations.
In 1975, Russel et al. (1) and Williamson and Fennel (2) showed that 4’,6-diamidino-2-phenylindole.2HCl (DAPI) reacts with DNA to form a fluorescent complex. They proposed that this compound could make DNA visible in cells infected with mycoplasma and/or viruses. There are bases for assumption that DAPI exhibits a preference for several continuous A-T base pairs (3-5). After experimental confirmation of the above phenomenon (6,7) we decided to investigate the mechanism of the DAPIpolynucleotides interaction. The specific intercalation of DAPI to DNA, which does not occur between DAPI and RNA (8,9), has enabled us to develop a simple fluorimetric micromethod for DNA analysis. The method appeared to be much more sensitive than those previously published. The lowest DNA concentration which can be detected as a DAPI-DNA complex, under the conditions given below, is 5 X lo-lo g/ml. Other fluorescent complexes have been used previously (lo- 12); however, only one of the methods, that described by Hill and Whatley (10) employing the antibiotic Mithramycin, is really simple and specific for DNA, but its sensitivity is limited to 5 x 10e7 g of DNA/ml. MATERIALS
AND METHODS
Equipment. The excitation and fluorescence spectra were obtained using an MPF-3 Hitachi-Perkin-Elmer spectrophotometer equipped with an R106 photomultiplier tube and a 150-W xenon lamp. Measurements of ’ To whom correspondence
should be addressed. 252
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 0003.2697
SIMPLE
AND
RAPID
DNA
MICROASSAY
253
DNA-DAPI complex were made at wavelengths of 372 and 454 nm, representing maxima of excitation and fluorescence, respectively. The fluorescence intensity of the examined sample, i,, was compared with is, the fluorescence intensity of Hitashi-Perkin-Elmer standard No. 4 in order to obtain reproducible results, independent of the width of the entrance and exit slits. The measurements of standard No. 4 were made at an excitation wavelength of 365 nm with equally wide slits. The relative fluorescence intensity has been expressed as i = (i,li& x 104. The sum of the relative fluorescence intensities of all components has been compared with i, (blank test). Measurements were made in l-cm--path silica cells using filter 39 to eliminate light dispersion. Reagents. DAPI was synthesized according to the procedure of Dann et al. (13) with modification (6); uv maxima = 224, 261, and 344 nm, and r = 23,500, 18,600, and 23,000, respectively. DAPI was dissolved in water to a concentration of 5.71 PM (2 pg/ml). This solution had an excitation maximum at wavelength 358 nm and a relative fluorescence intensity of i = 213.2 (when measured at the fluorescence maximum of 449 nm). Presently DAPI can be supplied by Serva Feinbiochemica, D.69 Heidelberg 1, P.O. Box 105260. Preparation of polynucleotides. The stock solution of DNA at a concentration of 250 pg/rnl was dissolved in 0.01 M NaCl (unless stated otherwise in the text). Dilutions of this DNA stock solution were also made with 0.01 M NaCl. DNA denaturation was carried out using a fivefold diluted DNA solution buffered with 0.005 M Hepes, pH 7. The samples were heated for 10 min at 100°C and were immediately chilled in ice. The degradation of stock solution to yield a sheared DNA preparation was carried out by using either (i) an MSE ultrasonicator (30-set sonications were repeated four times at 30-set intervals) or (ii) expelling the DNA solution through syringe needle No. 27 (14). Chemicals. All chemicals used were of high commercial purity and were tested for the presence of fluorescent impurities. The relative fluorescence intensity of 0.015 M solutions was between 1.0 and 5.0. General procedure. The solution of DAPI in water was mixed with the DNA solution in 0.01 M NaCl or 0.0066 M Na,SO, and was buffered with Tris or Hepes, at a final concentration of 0.005 M, to pH 7. These samples were then ready for measurement immediately after mixing or could be stored refrigerated for several weeks without any change in their optical properties prior to estimation. RESULTS
AND DISCUSSION
Figure 1 presents a comparison of the excitation and fluorescence spectra of the mixture of DNA and DAPI with the spectra of a DAPI solu-
254
KAPUSCIfiSKI
AND SKOCZYLAS
15
?O(
300
400
500
20
2.5
30
600 ml
FIG. 1. The excitation spectrum: (la) DNA-DAPI complex, (lb) DAPI solution. The fluorescence spectrum: (2a) DNA-DAPI complex, (2b) DAPI solution. DNA concentration in samples la and 2a is 20 ).&ml. DAPI concentration in all samples is 2 &ml. Samples are in 0.012 M NaCl, 0.05 M Hepes, pH 7. The excitation spectrum measurements (the excitation slit: 1 nm) were made at a fluorescence wavelength of 454 nm (emission slit: 20 nm). The fluorescence spectrum measurements (emission slit: 2 nm) were made at an excitation wavelength of 372 nm (excitation slit: 6 nm). Recorder sensitivity: 10; scan speed: 150 nm/min; chart speed: 40 mm/min. Inset: The influence of [DNA]/[DAPI] = I< on W (fluorescence intensification of the complex). [DNA] = 143 PM (50 &ml). Samples are in 0.012 M NaCl, 0.005 M Hepes, pH 7.
tion. There is a distinct fluorescence intensification of the mixture compared to the fluorescence of the individual components (the DNA solution showed no significant fluorescence under these experimental conditions). The fluorescence intensification, W = (i - i,)I[DAPI], depends on the ratio of components, R = [DNA]/[DAPI]. The strongest fluorescence intensification was obtained when R > 100 nucleotides/l molecule of DAPI, which is shown in the inset to Fig. 1. Practically the whole quantity of DAPI forms the fluorescent complex with DNA in this range of concentrations. The value of fluorescent intensification ( W) is also a function of the absolute DNA concentration. The highest values of W are reached by increasing values of R, if the DNA concentration
SIMPLE AND RAPID DNA MICROASSAY
255
increases. In very diluted samples, however, the highest value of W is reached at R > 5. Therefore, perhaps unexpectedly, the more diluted the DNA and DAPI solutions, the easier the formation of the fluorescent complex. For analytical purposes, a range of [DNA]/[DAPI] ratios should be used within which the fluorescent intensification (W) does not reach the maximal value. Therefore, the range of R = 0.28-5.5 for DNA concentrations of 5 x lo-lo to 1 x 10-j g/ml has been used with four DAPI concentrations (Fig. 2a-d). These results show that this method is useful for quantitative DNA assays over a wide range of DNA concentrations above 0.5 &ml. The ranges of DNA concentrations used in Fig. 2b and c, namely 5-1000 @ml, seem to be the most advantageous, because of their almost linear dependence. Various conditions can influence the fluorescence intensity in this method, and these are discussed below. Salt Concentration Increasing the ionic strength of the solutions reduces the fluorescence of the DAPI-DNA complex (Na+, chloride, perchlorate, acetate, EDTA). For accuracy, the ionic strength of the solutions used should be below 0.1. The ions of bivalent metals such as 2 x 10e3 M Mg2+, 1 x 10e3 M
FIG. 2. The calibration curves for quantitative DNA assay in complex with DAPI at varying DAPI concentrations applied to four different ranges of DNA concentration. i is the relative fluorescence intensity (i = (iA&) x 104; see Results and Discussion). DNA concentration in rig/ml = 0.5-10, 5-100, 50-1000, and 500-10,000 in a-d, respectively. DAPI concentration in @ml = 2, 20, 200, and 2000 in a-d, respectively. The samples are in 0.012M NaCl, 0.005 M Hepes, pH 7.
256
KAPUSCINSKI
AND SKOCZYLAS
Mn2+, 1 x 1O-3 M Ca2+ and anions such as 1 x 10-l M citrates and 1 x 10d3 M HPOa2- reduce fluorescence by a factor of approximately 2 in relation to NaCl solution of the same ionic strength. However, the fluorescence increases significantly, by a factor of approximately 2, when SOa2- ions are substituted for Cl- ions. The most suitable buffers for the assay were Tris, Hepes, and EDTA. Dependence
of Fluorescence
Intensity
on DNA Structure
The fluorescence intensity is highest when DAPI is complexed with native highly polymerized DNA and decreases when DAPI is complexed with degraded or denaturated DNA (see Table 1). However, this method may be used to analyze all these forms of DNA, if individual calibration curves are made. TABLE COMPARISON
1
OF THE RELATIVE FLUORESCENCE INTENSITIES OF DIFFERENT THE WHOLE HISTONE FRACTION, NUCLEOTIDES, AND UREA COMPLEXED WITH DAPI”
KINDS
OF
DNA AND RNA,
i - i.
Materials
Calf thymus high-polymerized, type I” Native DNA Native DNA sheared Native DNA sonicated Denaturated DNA Calf thymus high-polymerized native DNA” Herring high-polymerized native DNAd Calf thymus degraded DNAe Herring degraded DNA, type IV* Yeast high-polymerized RNA, type XI* Yeast high-polymerized RNA’ Yeast degraded RNAe Calf thymus high-polymerized native DNA, type Ib +RNA (type XI),* 1:l (w/w) +RNA (type XI),* 1:20 (w/w) +Histone,’ 1:l (w/w) +Urea, 0.1 M Nucleotides: AMP,’ GMP,’ UMP,d CMP” (each) Deoxynucleotides: * d-AMP, d-GMP, d-CMP, d-TMP (each) a The relative fluorescence complex is regarded as 100%. b Sigma Chemical Co. c Serva. d Calbiochem. e BDH. f PGCH. 0 FLUKA. h REANAL.
intensity
of the high-polymerized
100 94 74 33 100 102 38 9 3 1 3 97 92 92 116
