ANALYTICAL BIOCHEMISTRY 93, 306-311 (1979)
A Method of DNA Quantitation for Localization of DNA in Metrizamide Gradients DEBRA L. PETERS 1 AND MICHAEL E . D A H M U S Department of Biochemistry and Biophysics, University of California, Davis, California 95616 Received August 22, 1978 A quantitative assay for DNA is described that involves the binding of the cytological stain, methyl green, to DNA. At pH 7.9, solutions containing free methyl green undergo complete fading whereas solutions containing DNA-bound methyl green retain color in proportion to the amount of DNA present. The procedure allows for the quantitation of DNA in the presence of urea, sucrose, EDTA, protein, dithiothreitol, metrizamide, and low-concentration salts. This method is also applicable to the quantitation of DNA in chromatin.
The most commonly used color reaction for the quantitation of DNA in solution is the diphenylamine method originally described by Dische (1) and modified by Burton (2). Although the diphenylamine method is quite sensitive, a number of agents, among which are metrizamide and protein, interfere with this reaction. To remove interfering agents, samples must be precipitated and thoroughly washed with perchloric acid, a procedure which is not only time consuming but can result in significant loss of DNA. The method described in this paper is based on the DNA-binding property of the cytological stain, methyl green (3-5). The method is simple and is not subject to significant interference by metrizamide or a number of other common agents. These characteristics make methyl green a convenient reagent for localizing DNA in metrizamide gradients. MATERIALS AND METHODS
Reagents. Methyl green, color index No. 42590 (6), was purchased from Eastman Or-
ganic Chemicals. Tris, BSA, z SDS, and yeast RNA were obtained from Sigma Chemical Company. Ultrapure urea, sucrose, and (NH4)zSO4 were obtained from Schwarz/ Mann. Metrizamide was purchased from Nyegaard and Company, A/S, Oslo, and DTT was purchased from Calbiochem. Proteinase K was obtained from EM Laboratories. Preparations. Escherichia coli DNA was prepared by the method of Marmur (7) and rat liver and Novikoff ascites tumor DNA were prepared according to Dahmus and McConnell (8). Chromatin was prepared from Novikoff ascites cells as previously described (8). The concentration of DNA was estimated using Az60 = 20 cm 2 mg -1. RNA was dissolved in water to a final concentration of 1 mg/ml (estimated from A26o = 25 cm z mg-1). Methyl green solutions were made by dissolving the indicated amount of methyl green into 0.01 M Tris-HC1, pH 7.9. Trace amounts of crystal violet were removed by extraction with an equal volume of chloro-
1 Present address: Department of Plant Biology, Carnegie Institution of Washington, Stanford, Calif. 94305. 0003 -2697/79/040306-06502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any form reserved.
306
z Abbreviations used: BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; DTT, dithiothreitol, TCA, trichloroacetic acid; PCA, perchloric acid.
D N A Q U A N T I T A T I O N IN M E T R I Z A M I D E
form. Due to the rapid fading of methyl green at pH 7.9 (9), solutions cannot be stored as such and must be prepared just prior to use. General assay procedure. Unless otherwise stated the assay solutions were prepared by thoroughly mixing 0.2 ml of DNA solution with 1 ml of 0.01% methyl green solution. The solutions were incubated at 45°C in the dark for 3 h and the absorbance at 640 nm was determined. DNA concentration was estimated by comparison to a standard curve determined under the same buffer conditions. Chromatin samples were assayed following incubation with 50 ~g/ml proteinase K for 15 min at 37°. Metrizamide gradients. Metrizamide was dissolved in 10 mM Tris-HC1, pH 7.9, 1 mM EDTA to give final concentrations of 20 or 50%. An 8-ml linear gradient from 20-50% metrizamide was preformed and a 1-ml solution containing Novikoff ascites tumor chromatin was layered on top of the gradients. 2.0
15
o ~.D
1.0
L_
c o o
0
I
I
I
I
I
2
3
4
Time (hrs) FIG. 1. Time course of methyl green fading. Aliquots o f 0.2 ml containing no D N A (@), 20/xg D N A (A), or 60/zg D N A (©) were added to 1 ml of 0.01% methyl green solution and incubated at 45°C in a Gilford recording spectrophotometer. The absorbance at 640 n m was determined at the indicated times using 0.01 M T r i s HC1, p H 7.9, as a reference.
307
Samples were centrifuged in the Spinco 65 rotor at 50,000 rpm for 24 h at 4°C. Fractions of 0.35 ml were collected by inserting a capillary tube to the bottom of the gradient and removing the solution with a peristaltic pump. Aliquots of 0.05 ml were removed from each fraction, incubated with proteinase K, and assayed as described above. For determination of DNA by diphenylamine 0.1-ml aliquots were removed, precipitated by the addition of 0.025 ml 50% TCA, and after 2 h at 4° the pellets collected by centrifugation at 9400 g for 15 min. The pellets were washed with 5% PCA, hydrolyzed at 100°C, and assayed as described by Burton (2). Density was calculated from refractive index measurements using the relationship/9 = 3.350(c) - 3.462 [Ref. (9)]. RESULTS
Quantitation of DNA by Methyl Green Binding At pH 7.9 methyl green solutions that do not contain DNA undergo complete fading whereas solutions that contain DNA retain color in proportion to the amount of DNA present. It has been postulated that the fading is due to the conversion of the quinonoid form of the acyl to the benzenoid form with the formation of the carbinol (5). The time course of methyl green fading at 45°C in the presence of various concentrations of Novikoff ascites DNA is shown in Fig. 1. It is also possible to achieve adequate fading by incubating the sample at room temperature for 19 h. Tubes containing known amounts of DNA and a blank containing methyl green but no DNA were incubated as described under Methods (Fig. 2). When 1 ml of 0.01% methyl green solution is employed the reaction is linear with respect to DNA concentration to concentrations approaching 300 ~g. If, after the proper incubation time, the methyl green color is too intense for direct measurement the solution may be diluted with water and
308
PETERS AND DAHMUS
o 5.0
4.0 E 0 tD
3.0
3 o 2.0 JQ ~I
0.2
1,0
0.1
0 2 4 6 8 I
0
I00 /~g
I 200
I0 I
300
DNA
FIG. 2. Linearity of methyl green assay. Aliquots of 0.2 ml containing variable amounts of Novikoff ascites DNA were added to 1 ml of 0.01% methyl green solution. Absorbancies at 640 nm were read against a methyl green blank. All samples, except those in the insert, were diluted with 1 vol of water prior to the determination of A640. Absorbance measurements of diluted samples were then multiplied by 2 and plotted against DNA concentration.
tested, significantly interfere with the methyl g r e e n - D N A interaction (Table 1). Although MgC12 depresses the reactivity of methyl green with D N A , a standard curve supplemented with the appropriate amount o f salt can be used for the quantitation of D N A . The presence of metrizamide prevents the complete fading of methyl green in solutions containing no D N A and slightly decreased the color yield o f solutions containing D N A (Table 2). Since free D N A and nucleoprotein c o m p l e x e s band at densities ranging f r o m a b o u t 1.12-1.21 (9,12), the error involved in estimating D N A concentration should not be greater than 10%. I f the samples are read against a blank containing no metrizamide the incomplete fading of free methyl green nearly c o m p e n s a t e s for the decrease in binding resulting in an estimate that is only several percent lower than the actual concentration.
2.0
1.5 E
t h e A 6 4 0 determined without significantly altering the linearity of the assay. The standard curve for a variety o f D N A s , including poly(dAT), is shown in Fig. 3. The color d e v e l o p m e n t for a given D N A sample is highly reproducible. The slight variations observed between the different D N A s tested m a y be due to variations in A T content (10) and/or the presence of small amounts of denatured D N A (11). Denatured D N A shows a m a r k e d decrease in affinity for methyl green (11). We have o b s e r v e d a 60% reduction in the final A640 in the p r e s e n c e of heatdenatured DNA.
Effects of Various Agents on DN A Quantitation N e i t h e r sucrose, urea, NaC1, D T T , E D T A , R N A , nor BSA, in the amounts
O ,¢ t.O
o~
r~
io
t'~
0.5
0
I
I
I
I
l
20
40
60
80
I00
p.g DNA
FIG. 3. Standard curve for a variety of DNAs. Assay conditions are as described in the legend to Fig. 1 and under Methods except that the pea DNA assay was developed for 19 b at room temperature. Novikoff ascites DNA ([~), calf thymus DNA (©), rat liver DNA (A), pea DNA (0), E. coil DNA (X), and poly(dAT) (v).
DNA QUANTITATION IN METRIZAMIDE TABLE 1 EFFECT OF VARIOUSAGENTS ON DNA QUANTITATIONa Absorbance 640 nm Agentb
- DNA c
+ DNA
Control 2.4 M Sucrose 5 M Urea 2.0 M NaC1 5 mM MgC12 1 mM DTT 2 mM EDTA 100/zg/ml RNA 1 mg/ml BSA 1% SDS
0 0.068 0.024 0.018 0.003 0.008 0.059 0.013 0.016 >5
1.891 1.987 1.831 1.961 1.311 2.191 2.127 1.980 1.946 >5
Estimated DNA concentration (/zg)a 100 101 95.6 103 69.2 115 109 104 102
Each reaction contained 100/zg Novikoff ascites DNA. b The concentration refers to concentration in the DNA sample used for assay. c All samples were read against a 0.01 M Tris-HC1, methyl green blank. d Concentration was estimated during the standard curve run in the presence of 0.01 M Tris, pH 7.9. A more accurate estimate of DNA concentration can be made by utilizing a standard curve run in the same buffer. Of the reagents tested, the presence of S D S is c o m p l e t e l y i n c o m p a t i b l e w i t h this system. Although not tested, other acidic h i g h - m o l e c u l a r w e i g h t p o l y m e r s s u c h as lignin a n d c a r t i l a g e m a y i n t e r f e r e w i t h this assay.
Estimation of DNA Concentration in Chromatin A l t h o u g h B S A free in s o l u t i o n d o e s n o t a p p e a r to i n t e r f e r e w i t h e s t i m a t i o n o f D N A c o n c e n t r a t i o n , D N A - b o u n d p r o t e i n s d o interfere with the formation of a DNA-dye c o m p l e x . F o r this r e a s o n c h r o m a t i n s a m p l e s m u s t b e d e p r o t e i n i z e d p r i o r to D N A q u a n t i t a t i o n . A c o n v e n i e n t m e t h o d o f p r o t e i n rem o v a l is t h e i n c u b a t i o n o f c h r o m a t i n w i t h proteinase K. The DNA content of Novikoff ascites chromatin was determined by absorbance
309
at 260 n m in t h e p r e s e n c e o f 1% s o d i u m lauryl sarcosine, the diphenylamine method, and methyl green binding with and without treatment with proteinase K. A comparison o f t h e s e m e t h o d s is s h o w n in T a b l e 3. T h e r e is g o o d a g r e e m e n t b e t w e e n t h e d e t e r m i n a t i o n s b y A260 a n d m e t h y l g r e e n b i n d i n g following proteinase K digestion. The lower value determined by the diphenylamine m e t h o d m a y b e d u e t o l o s s e s in D N A d u r i n g t h e p r e c i p i t a t i o n a n d w a s h i n g p r i o r to a s s a y . The determination by methyl green without first r e m o v i n g c h r o m o s o m a l p r o t e i n s a l s o r e s u l t s in a n u n d e r e s t i m a t e of DNA concentration.
Detection of DNA in Metrizamide Gradients T h e u s e o f this a s s a y f o r d e t e c t i o n o f D N A in m e t r i z a m i d e g r a d i e n t s is d e m o n s t r a t e d in F i g . 4. N o v i k o f f a s c i t e s t u m o r c h r o m a t i n w a s b a n d e d in m e t r i z a m i d e a n d t h e D N A localized by the methyl green assay following d i g e s t i o n w i t h p r o t e i n a s e K , a n d t h e diphenylamine method. Both methods localize t h e D N A in t h e s a m e f r a c t i o n s o f t h e gradient although the actual concentration TABLE 2 EFFECT OF METRIZAMIDEON DNA QUANTITATION Absorbance 640 nm Percentage metrizamidea
-DNA
+DNA
Estimated DNA concentration (txg)b
0 20 40 60 80
0 0.022 0.045 0.067 0.079
1.320 1.280 1.231 1.187 1.119
100 95.3 89.8 84.8 78.8
The percentage metrizamide refers to the equivalent concentration in the 0.2-ml sample of DNA used for assay. For this experiment 0.5 ml of a 0.02% methyl green solution was added to 0.7 ml of a solution containing 100 t~g Novikoff ascites DNA and variable amounts of metrizamide. b The concentration of DNA was estimated after subtraction of the appropriate blank.
310
PETERS AND DAHMUS
within a given fraction varies with the method employed. The methyl green assay i n d i c a t e s t h e e n t i r e p e a k c o n t a i n s a b o u t 600 /zg D N A w h e r e a s t h e d i p h e n y l a m i n e a s s a y i n d i c a t e s the p e a k c o n t a i n s o n l y a b o u t 370 p~g D N A . A t o t a l o f 1.04 m g o f D N A w a s a p p l i e d to t h e g r a d i e n t .
DISCUSSION A l t h o u g h methyl green has b e e n u s e d quite e x t e n s i v e l y f o r t h e c y t o l o g i c a l staining o f n u c l e i a n d h a s b e e n s h o w n to b i n d to D N A (3,5,10), its u s e f o r d e t e c t i o n o f D N A in s o l u t i o n h a s n o t b e e n e x p l o i t e d . This p a p e r describes a method for easy detection of D N A in m e t r i z a m i d e g r a d i e n t s a n d is a p p l i c a b l e t o t h e q u a n t i t a t i o n o f D N A in c h r o m a t i n a n d a v a r i e t y o f s o l u t i o n s . S o m e o f the m o r e i m p o r t a n t a s p e c t s o f t h e a s s a y a r e its s e n s i t i v i t y , e a s e o f e x e c u t i o n , a n d ins e n s i t i v i t y to a v a r i e t y o f s u b s t a n c e s t h a t interfere with other DNA determinations. L i k e its d i p h e n y l a m i n e c o u n t e r p a r t , t h e
400
.9 o
300
o
1.5
o2OO, oo,
TABLE 3 QUANTITATION OF D N A IN CHROMATIN a
Method A260b
10.6
30.0
92.7
Methyl greenc Control Proteinase K Diphenylamine
7.8 10.0 7.8
18.5 28.6 20.8
63.0 98.6 70.0
Samples were prepared as described in the text. b Chromatin was adjusted to 1% sodium lauryl sarcosine and the absorbance measured at 260 nm. DNA was estimated using A260 = 20 cm2 mg -1. e Chromatin was assayed with and without treatment with proteinase K. The control sample was incubated for 15 min at 37°C in the presence of buffer and did not differ significantly from samples receiving no incubation. m e t h y l g r e e n a s s a y c a n d e t e c t as little as 2 / x g o f D N A in s o l u t i o n . T h e m e t h y l g r e e n a s s a y , h o w e v e r , has a g r e a t e r r a n g e . T h e reaction does not significantly deviate from l i n e a r i t y until t h e c o n c e n t r a t i o n o f D N A e x c e e d s 300/zg/0.2 ml. D u e to t h e l a c k o f significant i n t e r f e r e n c e by protein, urea, EDTA, and metrizamide, samples need not be routinely precipitated a n d w a s h e d p r i o r to a s s a y . T h e p r e s e n c e o f s o m e salt, e s p e c i a l l y MgC12, h o w e v e r resuits in t h e r e l e a s e o f m e t h y l g r e e n a n d m u s t b e c a r e f u l l y c o n t r o l l e d o r a v o i d e d . I n gene r a l h o w e v e r t h e m e t h o d p r o v i d e s a sensitive, reliable, and convenient assay for the quantitation of DNA under a variety of conditions.
,.2"3"',., o-o I0 Froction
20
1.0
Number
FIG. 4. Localization of DNA in metrizamide gradient. Novikoff ascites chromatin (1.04 mg in 1 ml) was applied to a 20-50% metrizamide gradient and centrifuged as described under Methods. Fractions were assayed for DNA by diphenylamine ([~) and methyl green binding (A).
DNA (/~g)
ACKNOWLEDGMENTS
We would like to thank Drs. George Bruening and Steve Daubert for their useful comments, suggestions, and the gift of methyl green. This work was supported in part by Grant HD-04899 from the United States Public Health Service, National Institutes of Health, awarded to M.E.D.D.L.P. was a predoctoral trainee of United States Public Health Service Training Grant GM 119 from the National Institutes of Health.
REFERENCES 1. Dische, Z. (1930) Mikrochemie 8, 4. 2. Burton, K. (1956)Biochem. J. 62, 315-323.
DNA QUANTITATION IN METRIZAMIDE 3. Kurnick, N. B. (1950)J. Gen. Physiol. 33,243-264. 4. Kurnick, N. B., and Herskowitz, I. H. (1952) J. Cell. Comp. Physiol. 39, 281-299. 5. Kurnick, N. B., and Foster, M. (1950)J. Gen. Physiol. 34, 147-159. 6. Lillie, R. (1969) in Conn's Biological Stains, Williams & Wilkins, Baltimore. 7. Marmur, J. (1961)J. Mol. Biol. 3, 208-218. 8. Dahmus, M. E., and McConnell, D. J. (1969) Biochemistry 8, 1524-1534.
31 1
9. Birnie, G. D., Rickwood, D., and Hell, A. (1973) Biochem. Biophys. Acta 331, 283-294. 10. Krey, A. K., and Hahn, F. E. (1975)Biochemistry 14, 5061-5067. 11. Scott, J. E., and WiUett, I. H. (1966) Nature (London) 209, 985-987. 12. Rickwood, D., and MacGillivray, A. J. (1977)Exp. Cell Res. 104, 287-292.
A Method of DNA Quantitation for Localization of DNA in Metrizamide Gradients DEBRA L. PETERS 1 AND MICHAEL E . D A H M U S Department of Biochemistry and Biophysics, University of California, Davis, California 95616 Received August 22, 1978 A quantitative assay for DNA is described that involves the binding of the cytological stain, methyl green, to DNA. At pH 7.9, solutions containing free methyl green undergo complete fading whereas solutions containing DNA-bound methyl green retain color in proportion to the amount of DNA present. The procedure allows for the quantitation of DNA in the presence of urea, sucrose, EDTA, protein, dithiothreitol, metrizamide, and low-concentration salts. This method is also applicable to the quantitation of DNA in chromatin.
The most commonly used color reaction for the quantitation of DNA in solution is the diphenylamine method originally described by Dische (1) and modified by Burton (2). Although the diphenylamine method is quite sensitive, a number of agents, among which are metrizamide and protein, interfere with this reaction. To remove interfering agents, samples must be precipitated and thoroughly washed with perchloric acid, a procedure which is not only time consuming but can result in significant loss of DNA. The method described in this paper is based on the DNA-binding property of the cytological stain, methyl green (3-5). The method is simple and is not subject to significant interference by metrizamide or a number of other common agents. These characteristics make methyl green a convenient reagent for localizing DNA in metrizamide gradients. MATERIALS AND METHODS
Reagents. Methyl green, color index No. 42590 (6), was purchased from Eastman Or-
ganic Chemicals. Tris, BSA, z SDS, and yeast RNA were obtained from Sigma Chemical Company. Ultrapure urea, sucrose, and (NH4)zSO4 were obtained from Schwarz/ Mann. Metrizamide was purchased from Nyegaard and Company, A/S, Oslo, and DTT was purchased from Calbiochem. Proteinase K was obtained from EM Laboratories. Preparations. Escherichia coli DNA was prepared by the method of Marmur (7) and rat liver and Novikoff ascites tumor DNA were prepared according to Dahmus and McConnell (8). Chromatin was prepared from Novikoff ascites cells as previously described (8). The concentration of DNA was estimated using Az60 = 20 cm 2 mg -1. RNA was dissolved in water to a final concentration of 1 mg/ml (estimated from A26o = 25 cm z mg-1). Methyl green solutions were made by dissolving the indicated amount of methyl green into 0.01 M Tris-HC1, pH 7.9. Trace amounts of crystal violet were removed by extraction with an equal volume of chloro-
1 Present address: Department of Plant Biology, Carnegie Institution of Washington, Stanford, Calif. 94305. 0003 -2697/79/040306-06502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any form reserved.
306
z Abbreviations used: BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; DTT, dithiothreitol, TCA, trichloroacetic acid; PCA, perchloric acid.
D N A Q U A N T I T A T I O N IN M E T R I Z A M I D E
form. Due to the rapid fading of methyl green at pH 7.9 (9), solutions cannot be stored as such and must be prepared just prior to use. General assay procedure. Unless otherwise stated the assay solutions were prepared by thoroughly mixing 0.2 ml of DNA solution with 1 ml of 0.01% methyl green solution. The solutions were incubated at 45°C in the dark for 3 h and the absorbance at 640 nm was determined. DNA concentration was estimated by comparison to a standard curve determined under the same buffer conditions. Chromatin samples were assayed following incubation with 50 ~g/ml proteinase K for 15 min at 37°. Metrizamide gradients. Metrizamide was dissolved in 10 mM Tris-HC1, pH 7.9, 1 mM EDTA to give final concentrations of 20 or 50%. An 8-ml linear gradient from 20-50% metrizamide was preformed and a 1-ml solution containing Novikoff ascites tumor chromatin was layered on top of the gradients. 2.0
15
o ~.D
1.0
L_
c o o
0
I
I
I
I
I
2
3
4
Time (hrs) FIG. 1. Time course of methyl green fading. Aliquots o f 0.2 ml containing no D N A (@), 20/xg D N A (A), or 60/zg D N A (©) were added to 1 ml of 0.01% methyl green solution and incubated at 45°C in a Gilford recording spectrophotometer. The absorbance at 640 n m was determined at the indicated times using 0.01 M T r i s HC1, p H 7.9, as a reference.
307
Samples were centrifuged in the Spinco 65 rotor at 50,000 rpm for 24 h at 4°C. Fractions of 0.35 ml were collected by inserting a capillary tube to the bottom of the gradient and removing the solution with a peristaltic pump. Aliquots of 0.05 ml were removed from each fraction, incubated with proteinase K, and assayed as described above. For determination of DNA by diphenylamine 0.1-ml aliquots were removed, precipitated by the addition of 0.025 ml 50% TCA, and after 2 h at 4° the pellets collected by centrifugation at 9400 g for 15 min. The pellets were washed with 5% PCA, hydrolyzed at 100°C, and assayed as described by Burton (2). Density was calculated from refractive index measurements using the relationship/9 = 3.350(c) - 3.462 [Ref. (9)]. RESULTS
Quantitation of DNA by Methyl Green Binding At pH 7.9 methyl green solutions that do not contain DNA undergo complete fading whereas solutions that contain DNA retain color in proportion to the amount of DNA present. It has been postulated that the fading is due to the conversion of the quinonoid form of the acyl to the benzenoid form with the formation of the carbinol (5). The time course of methyl green fading at 45°C in the presence of various concentrations of Novikoff ascites DNA is shown in Fig. 1. It is also possible to achieve adequate fading by incubating the sample at room temperature for 19 h. Tubes containing known amounts of DNA and a blank containing methyl green but no DNA were incubated as described under Methods (Fig. 2). When 1 ml of 0.01% methyl green solution is employed the reaction is linear with respect to DNA concentration to concentrations approaching 300 ~g. If, after the proper incubation time, the methyl green color is too intense for direct measurement the solution may be diluted with water and
308
PETERS AND DAHMUS
o 5.0
4.0 E 0 tD
3.0
3 o 2.0 JQ ~I
0.2
1,0
0.1
0 2 4 6 8 I
0
I00 /~g
I 200
I0 I
300
DNA
FIG. 2. Linearity of methyl green assay. Aliquots of 0.2 ml containing variable amounts of Novikoff ascites DNA were added to 1 ml of 0.01% methyl green solution. Absorbancies at 640 nm were read against a methyl green blank. All samples, except those in the insert, were diluted with 1 vol of water prior to the determination of A640. Absorbance measurements of diluted samples were then multiplied by 2 and plotted against DNA concentration.
tested, significantly interfere with the methyl g r e e n - D N A interaction (Table 1). Although MgC12 depresses the reactivity of methyl green with D N A , a standard curve supplemented with the appropriate amount o f salt can be used for the quantitation of D N A . The presence of metrizamide prevents the complete fading of methyl green in solutions containing no D N A and slightly decreased the color yield o f solutions containing D N A (Table 2). Since free D N A and nucleoprotein c o m p l e x e s band at densities ranging f r o m a b o u t 1.12-1.21 (9,12), the error involved in estimating D N A concentration should not be greater than 10%. I f the samples are read against a blank containing no metrizamide the incomplete fading of free methyl green nearly c o m p e n s a t e s for the decrease in binding resulting in an estimate that is only several percent lower than the actual concentration.
2.0
1.5 E
t h e A 6 4 0 determined without significantly altering the linearity of the assay. The standard curve for a variety o f D N A s , including poly(dAT), is shown in Fig. 3. The color d e v e l o p m e n t for a given D N A sample is highly reproducible. The slight variations observed between the different D N A s tested m a y be due to variations in A T content (10) and/or the presence of small amounts of denatured D N A (11). Denatured D N A shows a m a r k e d decrease in affinity for methyl green (11). We have o b s e r v e d a 60% reduction in the final A640 in the p r e s e n c e of heatdenatured DNA.
Effects of Various Agents on DN A Quantitation N e i t h e r sucrose, urea, NaC1, D T T , E D T A , R N A , nor BSA, in the amounts
O ,¢ t.O
o~
r~
io
t'~
0.5
0
I
I
I
I
l
20
40
60
80
I00
p.g DNA
FIG. 3. Standard curve for a variety of DNAs. Assay conditions are as described in the legend to Fig. 1 and under Methods except that the pea DNA assay was developed for 19 b at room temperature. Novikoff ascites DNA ([~), calf thymus DNA (©), rat liver DNA (A), pea DNA (0), E. coil DNA (X), and poly(dAT) (v).
DNA QUANTITATION IN METRIZAMIDE TABLE 1 EFFECT OF VARIOUSAGENTS ON DNA QUANTITATIONa Absorbance 640 nm Agentb
- DNA c
+ DNA
Control 2.4 M Sucrose 5 M Urea 2.0 M NaC1 5 mM MgC12 1 mM DTT 2 mM EDTA 100/zg/ml RNA 1 mg/ml BSA 1% SDS
0 0.068 0.024 0.018 0.003 0.008 0.059 0.013 0.016 >5
1.891 1.987 1.831 1.961 1.311 2.191 2.127 1.980 1.946 >5
Estimated DNA concentration (/zg)a 100 101 95.6 103 69.2 115 109 104 102
Each reaction contained 100/zg Novikoff ascites DNA. b The concentration refers to concentration in the DNA sample used for assay. c All samples were read against a 0.01 M Tris-HC1, methyl green blank. d Concentration was estimated during the standard curve run in the presence of 0.01 M Tris, pH 7.9. A more accurate estimate of DNA concentration can be made by utilizing a standard curve run in the same buffer. Of the reagents tested, the presence of S D S is c o m p l e t e l y i n c o m p a t i b l e w i t h this system. Although not tested, other acidic h i g h - m o l e c u l a r w e i g h t p o l y m e r s s u c h as lignin a n d c a r t i l a g e m a y i n t e r f e r e w i t h this assay.
Estimation of DNA Concentration in Chromatin A l t h o u g h B S A free in s o l u t i o n d o e s n o t a p p e a r to i n t e r f e r e w i t h e s t i m a t i o n o f D N A c o n c e n t r a t i o n , D N A - b o u n d p r o t e i n s d o interfere with the formation of a DNA-dye c o m p l e x . F o r this r e a s o n c h r o m a t i n s a m p l e s m u s t b e d e p r o t e i n i z e d p r i o r to D N A q u a n t i t a t i o n . A c o n v e n i e n t m e t h o d o f p r o t e i n rem o v a l is t h e i n c u b a t i o n o f c h r o m a t i n w i t h proteinase K. The DNA content of Novikoff ascites chromatin was determined by absorbance
309
at 260 n m in t h e p r e s e n c e o f 1% s o d i u m lauryl sarcosine, the diphenylamine method, and methyl green binding with and without treatment with proteinase K. A comparison o f t h e s e m e t h o d s is s h o w n in T a b l e 3. T h e r e is g o o d a g r e e m e n t b e t w e e n t h e d e t e r m i n a t i o n s b y A260 a n d m e t h y l g r e e n b i n d i n g following proteinase K digestion. The lower value determined by the diphenylamine m e t h o d m a y b e d u e t o l o s s e s in D N A d u r i n g t h e p r e c i p i t a t i o n a n d w a s h i n g p r i o r to a s s a y . The determination by methyl green without first r e m o v i n g c h r o m o s o m a l p r o t e i n s a l s o r e s u l t s in a n u n d e r e s t i m a t e of DNA concentration.
Detection of DNA in Metrizamide Gradients T h e u s e o f this a s s a y f o r d e t e c t i o n o f D N A in m e t r i z a m i d e g r a d i e n t s is d e m o n s t r a t e d in F i g . 4. N o v i k o f f a s c i t e s t u m o r c h r o m a t i n w a s b a n d e d in m e t r i z a m i d e a n d t h e D N A localized by the methyl green assay following d i g e s t i o n w i t h p r o t e i n a s e K , a n d t h e diphenylamine method. Both methods localize t h e D N A in t h e s a m e f r a c t i o n s o f t h e gradient although the actual concentration TABLE 2 EFFECT OF METRIZAMIDEON DNA QUANTITATION Absorbance 640 nm Percentage metrizamidea
-DNA
+DNA
Estimated DNA concentration (txg)b
0 20 40 60 80
0 0.022 0.045 0.067 0.079
1.320 1.280 1.231 1.187 1.119
100 95.3 89.8 84.8 78.8
The percentage metrizamide refers to the equivalent concentration in the 0.2-ml sample of DNA used for assay. For this experiment 0.5 ml of a 0.02% methyl green solution was added to 0.7 ml of a solution containing 100 t~g Novikoff ascites DNA and variable amounts of metrizamide. b The concentration of DNA was estimated after subtraction of the appropriate blank.
310
PETERS AND DAHMUS
within a given fraction varies with the method employed. The methyl green assay i n d i c a t e s t h e e n t i r e p e a k c o n t a i n s a b o u t 600 /zg D N A w h e r e a s t h e d i p h e n y l a m i n e a s s a y i n d i c a t e s the p e a k c o n t a i n s o n l y a b o u t 370 p~g D N A . A t o t a l o f 1.04 m g o f D N A w a s a p p l i e d to t h e g r a d i e n t .
DISCUSSION A l t h o u g h methyl green has b e e n u s e d quite e x t e n s i v e l y f o r t h e c y t o l o g i c a l staining o f n u c l e i a n d h a s b e e n s h o w n to b i n d to D N A (3,5,10), its u s e f o r d e t e c t i o n o f D N A in s o l u t i o n h a s n o t b e e n e x p l o i t e d . This p a p e r describes a method for easy detection of D N A in m e t r i z a m i d e g r a d i e n t s a n d is a p p l i c a b l e t o t h e q u a n t i t a t i o n o f D N A in c h r o m a t i n a n d a v a r i e t y o f s o l u t i o n s . S o m e o f the m o r e i m p o r t a n t a s p e c t s o f t h e a s s a y a r e its s e n s i t i v i t y , e a s e o f e x e c u t i o n , a n d ins e n s i t i v i t y to a v a r i e t y o f s u b s t a n c e s t h a t interfere with other DNA determinations. L i k e its d i p h e n y l a m i n e c o u n t e r p a r t , t h e
400
.9 o
300
o
1.5
o2OO, oo,
TABLE 3 QUANTITATION OF D N A IN CHROMATIN a
Method A260b
10.6
30.0
92.7
Methyl greenc Control Proteinase K Diphenylamine
7.8 10.0 7.8
18.5 28.6 20.8
63.0 98.6 70.0
Samples were prepared as described in the text. b Chromatin was adjusted to 1% sodium lauryl sarcosine and the absorbance measured at 260 nm. DNA was estimated using A260 = 20 cm2 mg -1. e Chromatin was assayed with and without treatment with proteinase K. The control sample was incubated for 15 min at 37°C in the presence of buffer and did not differ significantly from samples receiving no incubation. m e t h y l g r e e n a s s a y c a n d e t e c t as little as 2 / x g o f D N A in s o l u t i o n . T h e m e t h y l g r e e n a s s a y , h o w e v e r , has a g r e a t e r r a n g e . T h e reaction does not significantly deviate from l i n e a r i t y until t h e c o n c e n t r a t i o n o f D N A e x c e e d s 300/zg/0.2 ml. D u e to t h e l a c k o f significant i n t e r f e r e n c e by protein, urea, EDTA, and metrizamide, samples need not be routinely precipitated a n d w a s h e d p r i o r to a s s a y . T h e p r e s e n c e o f s o m e salt, e s p e c i a l l y MgC12, h o w e v e r resuits in t h e r e l e a s e o f m e t h y l g r e e n a n d m u s t b e c a r e f u l l y c o n t r o l l e d o r a v o i d e d . I n gene r a l h o w e v e r t h e m e t h o d p r o v i d e s a sensitive, reliable, and convenient assay for the quantitation of DNA under a variety of conditions.
,.2"3"',., o-o I0 Froction
20
1.0
Number
FIG. 4. Localization of DNA in metrizamide gradient. Novikoff ascites chromatin (1.04 mg in 1 ml) was applied to a 20-50% metrizamide gradient and centrifuged as described under Methods. Fractions were assayed for DNA by diphenylamine ([~) and methyl green binding (A).
DNA (/~g)
ACKNOWLEDGMENTS
We would like to thank Drs. George Bruening and Steve Daubert for their useful comments, suggestions, and the gift of methyl green. This work was supported in part by Grant HD-04899 from the United States Public Health Service, National Institutes of Health, awarded to M.E.D.D.L.P. was a predoctoral trainee of United States Public Health Service Training Grant GM 119 from the National Institutes of Health.
REFERENCES 1. Dische, Z. (1930) Mikrochemie 8, 4. 2. Burton, K. (1956)Biochem. J. 62, 315-323.
DNA QUANTITATION IN METRIZAMIDE 3. Kurnick, N. B. (1950)J. Gen. Physiol. 33,243-264. 4. Kurnick, N. B., and Herskowitz, I. H. (1952) J. Cell. Comp. Physiol. 39, 281-299. 5. Kurnick, N. B., and Foster, M. (1950)J. Gen. Physiol. 34, 147-159. 6. Lillie, R. (1969) in Conn's Biological Stains, Williams & Wilkins, Baltimore. 7. Marmur, J. (1961)J. Mol. Biol. 3, 208-218. 8. Dahmus, M. E., and McConnell, D. J. (1969) Biochemistry 8, 1524-1534.
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9. Birnie, G. D., Rickwood, D., and Hell, A. (1973) Biochem. Biophys. Acta 331, 283-294. 10. Krey, A. K., and Hahn, F. E. (1975)Biochemistry 14, 5061-5067. 11. Scott, J. E., and WiUett, I. H. (1966) Nature (London) 209, 985-987. 12. Rickwood, D., and MacGillivray, A. J. (1977)Exp. Cell Res. 104, 287-292.
