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Studies on the deproteinization of DNA
5o(i
B I O C I t l M I C A ET I2,1¢H'HYSICA ACTik
BBA 95595
S T U D I E S ON T H E D E P I ~ ( ) T E I N I Z A T I O N OF D N A
]afk, Calif. (U.S.A.)
(Received November 7th, ~966)
SUMMARY
The deproteinization of m a m m a l i a n D N A b y standard deproteinizing reagents (phenol, detergent, chloroform and organic salts) shows a pronounced time dependence which m a y a m o u n t to several days, if an o p t i n m m yield of D N A is desired. The physicoehemical properties of D N A obtained from extended extractions was not different from t h a t obtained from short-term extractions. It is concluded t h a t one of the difficulties experienced in extracting D N A from higher organisms m a y be due to inadequate periods of deproteinization.
INTRODUCTION
A general feature of methods used to prepare native, high molecular weight, low-protein D N A is their lack of universality when applied to a variety of tissues and organisms, particularly those of higher plants and animals 1,2. Difficulties have been attributed in large part to the diverse nature of D N A - b o u n d proteins and have p r o m p t e d the search for reagents effective in releasing D N A from certain tissues 1-a. This communication describes a procedure which helps to overcome the current disadvantages of having either to tailor an extraction procedure to a particular t y p e of tissue or being satisfied with a low yield of DNA. The procedure for D N A extraction reported here combines the methods of several workers2,< * with the modification t h a t enough time is allowed for m a x i m u m release of the DNA. Yields of up to 90 o;, of the D N A determined b y chemical analysis6, 7 have been obtained from nine different tissues representing two species. These included three types of hamster embryonic fibroblastic cells in culture: (I) normal secondary cells, (2) a p o l y o m a virus transformed cell line*, and (3) a spontaneously transformed cell line (G. FREEMAN, unpublished data). In addition, D N A wax preAbbreviation: SSC, o. 15 M NaCI, o.o15 M sodium citrate (pH 7.o); dilutions arc gixen as multiples. • 1~'. ( } R E E M A N , unpublished data cited by M. \rOOT AND 1<. I)ULBECCO, Proc. Natl. Acad. Sci. U.N., 46 (196o) 365 . Biochim. Biophys. Acta, 138 (1967) 506 512
STUDIES ON THE DEPROTEINIZATION OF
DNA
507
pared from cells cultured from a tumor induced in a newborn hamster by adenovirus type 7, adult hamster liver, human derived KB cells, human embryonic lung fibroblasts in their I l t h passage, and human embryonic lung cells in their 4oth passage after 'transformation' by infection with adenovirus-type 12 (ref. 8). Using phenol in conjunction with other reagents to prepare DNA from batches of tissue culture cells, more DNA was obtained from second and third extractions of the cellular debris than from the first extraction, provided that the extracting reagents were allowed to remain in contact with the debris for many hours or days. Subsequent kinetic studies indicated that the maximum release of DNA was obtained only after an extraction of many hours. This result is significant in view of the common 3o- or 6o-min extraction period used when phenol or other reagents are employed to liberate DNA, and may explain why low yields of high protein DNA are often obtained using a procedure not previously reported to have given a good yield for the tissue being studied. The deproteinization of human embryonic lung cells DNA was found to follow MICHAELIS--MENTEN9 kinetics in which the phenol and other extracting agents were considered catalysts (as a group}.
EXPERIMENTAL
DNA extraction procedure adopted All operations were carried out at room temperature (22-26°). The suspension of cells was washed by centrifuging at 15oo 250o rev./min for 7-1o rain; removing supernatant, and resuspending the cells in o.15 M NaC1 or suitable isotonic buffer. After removal of the supernatant the centrifuged cells were suspended in 0.3 M sodium triehloroacetate (pH 7.2) to give a eoncn, of 20-5o mg cells/ml, depending on the DNA content of the cells being studied; i.e., the DNA concentration was kept at 0.2-0.8 mg/ml (see ref. 4). Sodium dodecyl sulfate 25 % was added to give a final concn, of I.O %. After shaking for no more than a few minutes to lyse the cells, the suspension was shaken gently for several days with 0.2-0. 4 vol. of a (4 : I, v/v) mixture of redistilled phenol containing o.I 0'o 8-hydroxyquinoline to o.3 M sodium trichloroacetate. After the phases were separated by centrifugation the phenol extraction was at times repeated by shaking at least 24 h with additional phenol. After removal of the phenol, 0.2-0.4 vol. chloroform-octanol (24:1, v/v) was added, and the mixture was shaken gently for 30 min or longer. Additional chloroformoetanol extractions were performed until little protein was seen at the interface of the solutions. The DNA in the aqueous phase was then precipitated by layering 1.8-2.o volumes of 95 °o ethanol or absolute ethanol on the aqueous solution and spooling the DNA fibers on a glass stirring rod. Excess alcohol was removed by pressing the DNA against the side of the beaker. The precipitate was then dispersed in sufficient o.I ×SSC (SSC is o.15 M NaC1, o.o15 M sodium citrate (pH 7.0)) to give a DNA concentration of 0.2 0.8 rag/m1. After dissolution of the DNA the solution was brought to I ×SSC by addition of IO xSSC. Pancreatic ribonuclease (EC 2.7.7.I6 ), I mg/ml in o.15 M NaCl-o.I M EDTA (pH 8.o), was added to give a final conen, of 50/~g/ml. The ribonuclease was heated to 80 ° for IO min prior to use to inactivate possible deoxyribonuclease contaminants. After incubation for i h at 37 ° the solution was subjected to chloroformBiochim. Biophys. dcta, 138 (1967) 5o6-512
50~
R.S.
YOLLES, G. FREEMAN
I-octanol extractions until little or no protein was seen at the interface. The D N A was again precipitated with 1.8-2.o vol. ethanol, and the drained precipitate was dispersed in sufficient o.I ×SSC to give a D N A concn, of o.2-o.S mg/ml. To the dispersed precipitate was added o.I vol. 3.o M sodium acetate, o.ooi M in E D T A , and the D N A was precipitated by adding dropwise o.54 vol. 2-propanol while the D N A solution was being rapidly stirred. At about o.45-o.5 w~l. the bulk of the material started to precipitate and wind around the propeller unless the D N A concentration was low, in which case more 2-propanol was added. When precipitation began the solution was stirred more slowly and the 2-propanol added more slowly. The 2-propanol precipitation was repeated. The resulting precipitate was dissolved in o.I ×SSC and IO x S S C was added to bring the concentration to I ×SSC. Chloroform-I-octanol extractions were again performed until no visible protein was seen at the interface.
Kinelic studies Time-course studies were made by carrying out the extractions in screw cap bottles which were placed on a Burrell wrist-action shaker at a setting of 5 or less. At various times the extraction mixture was centrifuged at low speed to separate the phases and o.I- or o.2-ml aliquots were removed for analysis. An equal volume of o.3 M sodium trichloroacetate (pH 7.2) (with I ° o sodium dodecyl sulfate) was then added to maintain a constant aqueous volume. The procedure was carried out at room temperature (22 20°).
RESUI.TS AND DISCUSSION
Table I summarizes D N A yields with optical characteristics obtained from cell lines and tissues, according to the n u m b e r of hours the cellular debris remained in contact with the phenol and other reagents. The D N A extracted from an 'aged' batch of cells appeared to be of similar quality with respect to nativeness, protein, and guanine-cytosine content, because absorbance ratios, melting temperature, and percent hyperchromism were not significantly different for later extracts as compared with the initial extracts. B o t h early and late extractions gave D N A solutions t h a t were judged equally viscous. It was observed t h a t for the aged preparations the n u m b e r of chloroform-I-octanol deproteinization steps were usually reduced and the entire preparation proceeded more smoothly. The protein content of the D N A preparations can be estimated on the basis that a z6o mf~/z3o m# ratio of 2.2 corresponds to about o.3-o.5 °o protein 1°. The 26o m y 2 3 o m~t ratio is probably more significant than the often quoted 26o m/~/28o mi~ ratio, because the former reflects absorbance due to the amide group, present in all peptides, whereas the latter is sensitive only to the aromatic amino acids. Kinetic analysis of DNA-liberation from two cell lines (adenovirus-type 7 and h u m a n embryonic lung) are shown in Figs. I and 2. The results indicate t h a t m a n y hours or days of contact are necessary to obtain a high yield of DNA. These observations are consistent with the data plotted in Fig. 3, showing the yield of Biochim. Biophys. ~4cIa, ~38 (1967) 506 51z
'
i
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J
vo .q v
0o
%
t
DNA
20o 91o 12oo 270 65o 47 ° 4500 320 464 ° 80 420 2300 45 ° lO3O 850 12oo 250 186o
g cells
o/
5.6 25.0 33.0 18.8 45 5.2 50.0 3.6 51.o 2.6 13.5 74.0 12.6 29.0 > 5° 80.6 16.8 --
Theor.
o~ .o
extracted
ygDNAI
• P r e p a r a t i o n s u s e d in k i n e t i c s t u d i e s (see F i g s . I a n d 2).
i7 25- 3° 24 28 2 4 - 28 43 ° ; 7 17o 620 600 1 4 2 2 - 26 19o I 4 380 500 95 ]2o 12oo lOO
1 2 I i '2 i 2 3 i I 2 3 I 2 I I 2 I
Ha2°-I Ha2°-I H a 2 ° [aH~-I HaLiv-I HaLiv-I Ad7HaT II Ad7HaT-11 Ad7HaT-I[ Ad7HaT-Ill* ]?YTFHa[aHI-I PYTFHa[aHI-I P Y T F H a ~ 3 H J -I SPTFHa-I SPTFHa-1 HEL-I * ADI2TFHEL-I ADI2TFHEL-I 1,2B- [
(h)
Extraclion Contact with No. reagents
2"issue source
PHB, PHB, NSA 8HQ 8HQ 8HQ, 8HQ, 8HQ, 8HQ PHB PHB PHB PHB PHB, 8HQ 8HQ 8HQ 8HQ NSA
PHB PHB PHB
NSA NSA
Additional reagents z~sed
1.77 1.69 1.86 1.55 i .85 1.92 1.84 1.86 1.85 1.85 1-83 1.87 1.88 1.84 1.85 1.81 1.83 1.89
260/280
--2.11 1.24 1.91 2.66 2.35 2.36 2.36 -1.94 -1.78 2.30 2.44 2.25 2.20 2.36
260,/230
Absorbance ratios (mlt )
87.2 86. 7 86,0 87. 3 86.6 87.2 86.6 86.6 87. 4 _ 86.8 86.2 86. 3 86.6 87-2 84. 4 84.2 --
7"m ( ± o.8 ° )
36 28 39 19 3° 38 39 41 41 _ 35 36 38 4° 38 2>3 ° 39 --
°/o Hyperchromism
Thermal denaturation data
T h e f o l l o w i n g a b b r e v i a t i o n s for t h e cell l i n e s a n d t i s s u e s h a v e b e e n u s e d : H a , h a m s t e r ; H , h u m a n ; 2 °, s e c o n d a r y - c u l t u r e d f i b r o b l a s t s ; L i v , a d u l t l i v e r ; A d 7 a n d Ad12, a d e n o v i r u s - t y p e 7 a n d 12, r e s p e c t i v e l y ; P Y , p o l y o m a v i r u s ; T F , t r a n s f o r m e d ; T, t u m o r ; SP, s p o n t a n e o u s ; ( H ) E I . , ( h t m l a n ) e m b r y o n i c l u n g ; I¢B, h u m a n t,:B cells. All d e s i g n a t i o n s r e f e r t o c e l l s p r o p a g a t e d i n t i s s u e c u l t u r e e x c e p t for H a L i v ( a d u l t h a m s t e r l i v e r ) . T h e I { o m a n n u m e r a l r e f e r s to t h e c e l l b a t c h . To s o m e cell c u l t u r e s aH w a s a d d e d in t h e f o r m of I 3 l e - a H ~ t h y m i d i n e . A l l t h e extractions were carried out at room temperature using phenol, sodium trichloroacetate and sodium dodecvl sulfate. In addition some p r e p a r a t i o n s c o n t a i n e d o r g a n i c s a l t s a s i n d i c a t e d in t h e t a b l e : P H B , p - h y d r o x v b e n z o a t e ; N S A , 2 - n a p h t h a l e n e s u l f o n a t e ; 8 H Q , 8 - h y d r o x y q u i n o l i n e . T h e w e i g h t of t h e cells w a s e s t i m a t e d o n t h e b a s i s of t h e i r v o l u m e a f t e r c e n t r i f u g a t i o n a t 1 5 o o - 2 5 o o r e v . / m i n for i o 15 m i n , a s s u u l i n g a d e n s i t y of 1.1 g / m l . T h e y i e l d of D N A w a s b a s e d o n a n e x t i n c t i o n of 4 o / ~ g / A m l (1 c m p a t h - l e n g t h a t 26o m / , ) . T h e D N A c o n t e n t of t h e cells w a s d e t e r m i n e d b y t h e d i p h e n y l a m i n e r e a c t i o n of t3URTON 6 o r DXSCHE v. T h e m e l t i n g t e m p e r a t u r e (Tin) a n d % h y p e r c h r o m i s m w e r e d e t e r m i n e d in 1.o x SSC xs (no c o r r e c t i o n s w e r e m a d e for t h e r m a l e x p a n s i o n of t h e s o l u t i o n ) .
CHARACTERISTICS OF D N A PREPARATIONS
TABLE
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440
Fig. ~. A c t i o n of p h e n o l a n d o t h e r e x t r a c t i n g r e a g e n t s on t h e re l e a s e of D N A from h u m a n e n> b r y o n i c l u n g cells. To 2 g of t h e s e cells were a d d e d 15 ml o. 3 M s o d i u m t r i c h l o r o a c e t a t e (pH 7.2), I ~}{jin s o d i u m d o d e c y l s u l f a t e a n d 4 m l r e d i s t i l l e d phenol , o.i % in 8 - h y d r o x y q u i n o l i n e . O r d i n a t e is t h e c o n c e n t r a t i o n of D N A in t h e a q u e o u s phase. Th e r e a c t i o n w a s c a r r i e d o u t a t 23 26 °. E x p l a n a t i o n of a r r o w s : A (79 h), s o l u t i o n frozen for 24 h (time n o t i n c l u d e d on a bs c i s s a ); B (18~ h), o.25 vol. c h l o r o f o r n l - o c t a n o l (24 : i, v / v ) added. A t 226 h t h e a q u e o u s l a y e r w a s r e m o v e d a n d r e p l a c e d w i t h IO ml fresh s o d i u m t r i c h l o r o a c e t a t e - s o d i u m d o d e c y l sulfate. The c u r v e w a s d r a w n a c c o r d i n g to t h e e q u a t i o n g i v e n in t h e t e x t . I
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Fig. 2. A c t i o n of p h e n o l a n d e x t r a c t i n g r e a g e n t s on tile release of I ) N A from cells c u l t u r e d from a t u m o r i n d u c e d in a h a m s t e r by' a d e n o v i r u s - t y p e 7. To 1. 5 g of t he cells were a d d e d ~i. 5 ml o. 3 M s o d i u m t r i c h l o r o a c e t a t e (pH 7.2), I o~) in s o d i u m d o d e c y l s ul fa t e a nd i. 5 ml r e d i s t i l l e d phenol, o.I O,/o in 8-hvdroxvquinoline.. O r d i i m t e is t o t a l nag D N A re l e a s e d pe r g of cells. The r e a c t i o n was c a r r i e d o u t a t 23 26:. E x p l a n a t i o n of a r r o w s : A (248 h), o.43 vol. s odi ul n t ri c hl oroa c e t a t e s o d i u m d o d e c v l s u l f a t e a d d e d to soln.; B (297 h), a q u e o u s s o l u t i o n r e m o v e d a nd r e p l a c e d w i t h 7 ml s o d i u m t r i c i a l o r o a c e t a t e s o d i m n d o d e c v l s u l f a t e plus 2 ml phe nol ; C (315 h), a q u e o u s s o l u t i o n r e m o v e d aIxl c o m b i n e d w i t h the s o l u t i o n p r e v i o u s l y r e m o v e d a t 297 h; I) (345 h), o.25 vol. chloroforn: o c t a n o l ( 2 4 : t , v/v) added. Fig. 3. Yield of I)NA ol)tained from s e v e r a l t y p e s of tis~.ue as a f u n c t i o n of tile numl )e r of h o u r s t h e c e l l u l a r m a t e r i a l r e m a i n e d in c o n t a c t w i t h t h e e x t r a c t i n g r e a g e n t s . O, 1 lu 2"; ~ , H a l.iv; A, A d 7 H a t ; V , P Y T F H a ; O , S P T F Ha. The d a t a a n d a b b r ( v i a t i o n s for tile cell t y p e s are t a k e n from T a b l e 1. T o t a l I ) N A w a s d e t e r m i n e d l:y the d i p h e n y q a m i n e r e a c t i o n of I{URTON'i or I)ISCHIC7.
DNA from several cell types as a function of the hours of contact with the extracting reagents. The kinetics of deproteinization may be outlined by the following reaction steps: /"1 DNA-protein-~-R +~-~DNA protein R k,, Biochim. Biophys..tcta, 138 (1967) 506 512
(I)
STUDIES ON THE DEPROTEINIZATION OF DNA
511
k3
D N A - p r o t e i n - R ~ DNA +protein + R
(2)
k4 If
d[DNA-Pr-R] dt
= o (steady-state condition) and k4
o
(a reasonable
assumption because the bulk of the protein precipitates out of solution after separation from the DNA) one can apply the MICHAELIS-MENTEN1° equation:
d [DNA ] dt
k s ~R] --
i+
Km
(3)
[DN A-protein !
where [DNA ], [DNA-protein~ and [R l are the concentrations of DNA, DNA-protein and combined extracting reagents, respectively, and K m = (k2÷ka)/kl. Integrating, one obtains:
[-
~DNAIr
1
Km In ([DNA If-- [DNAJJ -- [DNA~ = ka[R]t
(4)
where [DNA ]r is the final concentration of DNA. For the deproteinization of human embryonic lung cell DNA I(m and ks have the values 34 °/,g/ml and 3.5" IO 6.h-1 respectively, where [DNAir is taken as 313/,g/ml and [RI ~ 800 mg/ml phenol. The good correspondence between the experimental points and Eqn. 4 as shown in Fig. I is consistent with the reaction steps outlined in Eqn. I and 2. It is noteworthy that the addition of various organic salts to the deproteinizing reagents, as shown in Table I and Fig. 3, did not change the general feature of the need for long periods of deproteinization in order to obtain maximum yields of DNA. There is a dearth of references to the kinetics of DNA extraction although several investigators have reported long-term DNA extractions. MIRSKY AND POLLISTER11 described a 48-h chloroform-octanol extraction at pH i o - i i for removal of DNA-bound protein (a condition which causes denaturation of DNA). DOUNCE AND MONTY12 reported that a 24-h stirring period in I.O M NaCI was needed before a satisfactory yield of calf-thymus DNA could be obtained by further treatment with sodium dodecyl sulfate, a result consistent with reference by CHARGAFF13 to a 4-day stirring period of calf-thymus nucleoprotein in saturated NaC1. The limitation of the solubility of the nucleoprotein as a factor in DNA release has been discussed by COLTER, BROWN" AND ELLEM 14, who employed phenol to extract DNA from mouse Ehrlieh aseites cells, mouse liver, and pneumocoecus. It is clear that better yields of DNA from the tissue types studied here can be obtained by allowing the DNA-extraeting reagents to remain in contact with the cells or tissue. How general this feature is will depend on the outcome of studies with other tissues. Optimum yields of DNA are important not only in terms of economy but because the possibility remains that the composition of extracted DNA may not faithfully represent the DNA in vivo if all or nearly all of the DNA is not extracted. Biases in the DNA composition could result if the procedure favored extraction of DNA with either a higher or lower G + C content, or DNA that is bound to certain proteins and not to others, e.g., repressed DNA as opposed to derepressed DNA. Such a consideration is important in view of the fact that a small Biochim. Biophys. Acta, 138 (1967) 5o6-512
512
17,. S. YOLLES, G. FREEMAN
f r a c t i o n of t h e D N A i n a d i f f e r e n t i a t e d cell is i n a d e r e p r e s s e d s t a t e a t a n y g i v e n t i m e 15. I t is l i k e l y t h a t t h e i n f l u e n c e of t i m e o n D N A d e p r o t e i n i z a t i o n h a s n o t b e e n a d e q u a t e l y s t u d i e d . Y e t t h e c h e m i s t r y of d e p r o t e i n i z a t i o n is a k e y t o u n d e r s t a n d i n g t h e n a t u r e of b o n d s i n t h e n u c l e o p r o t e i n c o m p l e x w h i c h , if f u l l y e l u c i d a t e d , c o u l d p r o v i d e i n f o r m a t i o n b a s i c t o t h e u n d e r s t a n d i n g of g e n e r e p r e s s i o n .
ACKNOWLEDGEMENTS
W e t h a n k D r . I. V. SULTANIAN for s u p p l y i n g t h e t i s s u e c u l t u r e cells a n d for h e l p f u l d i s c u s s i o n s . W e a r e p a r t i c u l a r l y i n d e b t e d t o Mr. LAWRENCE HOOSER, Mr. RICHARD J . JONES, a n d Mrs. AUDREY KUEHN for t h e i r e x c e l l e n t t e c h n i c a l a s s i s t a n c e a n d t o D r . F.-C. CHAO a n d Mrs. ANN GRIFFIN for s u p p l y i n g d a t a c o n c e r n i n g t h e t o t a l D N A c o n t e n t of s e v e r a l of t h e h a m s t e r cell lines. T h i s w o r k w a s s u p p o r t e d i n p a r t b y N a t i o n a l I n s t i t u t e s of H e a l t h G r a n t x S O I F R - o 5 5 2 2 f r o m t h e F a c i l i t i e s R e s o u r c e s B r a n c h a n d C o n t r a c t No. P H 4 3 - 6 5 - 4 3 , CCNSC, N C I .
]~EFERENCES I K. S. ]'~IRBY, JProgr. Exptl. Tumor Res., 2 (I96I) 29i. 2 1,2. S. 1,1IRBY, in J. N. DAVIDSON AND \V. g. COHN, Progress in NT~cleic Acid Research and Molecular Biology, Vol. 3, Academic Press, New York, 1964, p. I. 3 K. S. KIRBY, Biochenz. J., 66 (1957) 4954 J- MARMUR, in S. P. COLOWlCK AND N. O. NAPLAX, Methods in ]Snzyuzology, Vol. V[, Acadenlic Press, New York, 1963, p. 726. 5 P. M. B. -WALKER AND A. MCLAREN, .]. 3Iol. Biol., 12 (1965) 3946 K. BURTON, Biochem. J., 62 (1956 ) 315 . 7 Z. I)ISCHE, in E. CHARGAFF AND J. N. DAVII)SON, The Nucleic Acids, Vol. i, Academic Press, New York, I955, p. 285. 8 I. V. SULTANIAN AND G. lgREEMAN, Science, 154 (1966) 665. 9 L. ~{ICHAELIS AND M. L. MENTEN, Bioche~n. Z., 49 (1913) 333. 10 J. MARMUR, J. Mol. Biol., 3 (1961) 208. 11 A. E. MIRSKY AND A. "~\r. POLLISTER, ,]. Gen. Physiol., 3 ° (1946) 117. 12 A. L. 1)OUNCE AND K. J. MONTY, J. Biophys. Biochem. Cytol., 1 (J955) 15513 E. CHARC,AI~F, in E. CHARGAVF ANt) J. N. DaV]DSON, The Nucleic .tcids, Vol. I, Academic Press, New York, 1955, p. 3o7 . J4 J. S. CCmTER, 1¢. A. BROWN AND 1~. A. O. ELLEM, Biochim. Biophys...Icta, 55 (1962) 31. ]5 J. PAUL AND N. S. GILMOUR, .]. Mol. Biol., 16 (z966) 242. 16 J. MARMUR AND l ), DOTY, .[. Mol. Biol., 5 (]962) lO9. Biochim. Biophys. ~4cta, I3S (1967) 506 512
B I O C I t l M I C A ET I2,1¢H'HYSICA ACTik
BBA 95595
S T U D I E S ON T H E D E P I ~ ( ) T E I N I Z A T I O N OF D N A
]
(Received November 7th, ~966)
SUMMARY
The deproteinization of m a m m a l i a n D N A b y standard deproteinizing reagents (phenol, detergent, chloroform and organic salts) shows a pronounced time dependence which m a y a m o u n t to several days, if an o p t i n m m yield of D N A is desired. The physicoehemical properties of D N A obtained from extended extractions was not different from t h a t obtained from short-term extractions. It is concluded t h a t one of the difficulties experienced in extracting D N A from higher organisms m a y be due to inadequate periods of deproteinization.
INTRODUCTION
A general feature of methods used to prepare native, high molecular weight, low-protein D N A is their lack of universality when applied to a variety of tissues and organisms, particularly those of higher plants and animals 1,2. Difficulties have been attributed in large part to the diverse nature of D N A - b o u n d proteins and have p r o m p t e d the search for reagents effective in releasing D N A from certain tissues 1-a. This communication describes a procedure which helps to overcome the current disadvantages of having either to tailor an extraction procedure to a particular t y p e of tissue or being satisfied with a low yield of DNA. The procedure for D N A extraction reported here combines the methods of several workers2,< * with the modification t h a t enough time is allowed for m a x i m u m release of the DNA. Yields of up to 90 o;, of the D N A determined b y chemical analysis6, 7 have been obtained from nine different tissues representing two species. These included three types of hamster embryonic fibroblastic cells in culture: (I) normal secondary cells, (2) a p o l y o m a virus transformed cell line*, and (3) a spontaneously transformed cell line (G. FREEMAN, unpublished data). In addition, D N A wax preAbbreviation: SSC, o. 15 M NaCI, o.o15 M sodium citrate (pH 7.o); dilutions arc gixen as multiples. • 1~'. ( } R E E M A N , unpublished data cited by M. \rOOT AND 1<. I)ULBECCO, Proc. Natl. Acad. Sci. U.N., 46 (196o) 365 . Biochim. Biophys. Acta, 138 (1967) 506 512
STUDIES ON THE DEPROTEINIZATION OF
DNA
507
pared from cells cultured from a tumor induced in a newborn hamster by adenovirus type 7, adult hamster liver, human derived KB cells, human embryonic lung fibroblasts in their I l t h passage, and human embryonic lung cells in their 4oth passage after 'transformation' by infection with adenovirus-type 12 (ref. 8). Using phenol in conjunction with other reagents to prepare DNA from batches of tissue culture cells, more DNA was obtained from second and third extractions of the cellular debris than from the first extraction, provided that the extracting reagents were allowed to remain in contact with the debris for many hours or days. Subsequent kinetic studies indicated that the maximum release of DNA was obtained only after an extraction of many hours. This result is significant in view of the common 3o- or 6o-min extraction period used when phenol or other reagents are employed to liberate DNA, and may explain why low yields of high protein DNA are often obtained using a procedure not previously reported to have given a good yield for the tissue being studied. The deproteinization of human embryonic lung cells DNA was found to follow MICHAELIS--MENTEN9 kinetics in which the phenol and other extracting agents were considered catalysts (as a group}.
EXPERIMENTAL
DNA extraction procedure adopted All operations were carried out at room temperature (22-26°). The suspension of cells was washed by centrifuging at 15oo 250o rev./min for 7-1o rain; removing supernatant, and resuspending the cells in o.15 M NaC1 or suitable isotonic buffer. After removal of the supernatant the centrifuged cells were suspended in 0.3 M sodium triehloroacetate (pH 7.2) to give a eoncn, of 20-5o mg cells/ml, depending on the DNA content of the cells being studied; i.e., the DNA concentration was kept at 0.2-0.8 mg/ml (see ref. 4). Sodium dodecyl sulfate 25 % was added to give a final concn, of I.O %. After shaking for no more than a few minutes to lyse the cells, the suspension was shaken gently for several days with 0.2-0. 4 vol. of a (4 : I, v/v) mixture of redistilled phenol containing o.I 0'o 8-hydroxyquinoline to o.3 M sodium trichloroacetate. After the phases were separated by centrifugation the phenol extraction was at times repeated by shaking at least 24 h with additional phenol. After removal of the phenol, 0.2-0.4 vol. chloroform-octanol (24:1, v/v) was added, and the mixture was shaken gently for 30 min or longer. Additional chloroformoetanol extractions were performed until little protein was seen at the interface of the solutions. The DNA in the aqueous phase was then precipitated by layering 1.8-2.o volumes of 95 °o ethanol or absolute ethanol on the aqueous solution and spooling the DNA fibers on a glass stirring rod. Excess alcohol was removed by pressing the DNA against the side of the beaker. The precipitate was then dispersed in sufficient o.I ×SSC (SSC is o.15 M NaC1, o.o15 M sodium citrate (pH 7.0)) to give a DNA concentration of 0.2 0.8 rag/m1. After dissolution of the DNA the solution was brought to I ×SSC by addition of IO xSSC. Pancreatic ribonuclease (EC 2.7.7.I6 ), I mg/ml in o.15 M NaCl-o.I M EDTA (pH 8.o), was added to give a final conen, of 50/~g/ml. The ribonuclease was heated to 80 ° for IO min prior to use to inactivate possible deoxyribonuclease contaminants. After incubation for i h at 37 ° the solution was subjected to chloroformBiochim. Biophys. dcta, 138 (1967) 5o6-512
50~
R.S.
YOLLES, G. FREEMAN
I-octanol extractions until little or no protein was seen at the interface. The D N A was again precipitated with 1.8-2.o vol. ethanol, and the drained precipitate was dispersed in sufficient o.I ×SSC to give a D N A concn, of o.2-o.S mg/ml. To the dispersed precipitate was added o.I vol. 3.o M sodium acetate, o.ooi M in E D T A , and the D N A was precipitated by adding dropwise o.54 vol. 2-propanol while the D N A solution was being rapidly stirred. At about o.45-o.5 w~l. the bulk of the material started to precipitate and wind around the propeller unless the D N A concentration was low, in which case more 2-propanol was added. When precipitation began the solution was stirred more slowly and the 2-propanol added more slowly. The 2-propanol precipitation was repeated. The resulting precipitate was dissolved in o.I ×SSC and IO x S S C was added to bring the concentration to I ×SSC. Chloroform-I-octanol extractions were again performed until no visible protein was seen at the interface.
Kinelic studies Time-course studies were made by carrying out the extractions in screw cap bottles which were placed on a Burrell wrist-action shaker at a setting of 5 or less. At various times the extraction mixture was centrifuged at low speed to separate the phases and o.I- or o.2-ml aliquots were removed for analysis. An equal volume of o.3 M sodium trichloroacetate (pH 7.2) (with I ° o sodium dodecyl sulfate) was then added to maintain a constant aqueous volume. The procedure was carried out at room temperature (22 20°).
RESUI.TS AND DISCUSSION
Table I summarizes D N A yields with optical characteristics obtained from cell lines and tissues, according to the n u m b e r of hours the cellular debris remained in contact with the phenol and other reagents. The D N A extracted from an 'aged' batch of cells appeared to be of similar quality with respect to nativeness, protein, and guanine-cytosine content, because absorbance ratios, melting temperature, and percent hyperchromism were not significantly different for later extracts as compared with the initial extracts. B o t h early and late extractions gave D N A solutions t h a t were judged equally viscous. It was observed t h a t for the aged preparations the n u m b e r of chloroform-I-octanol deproteinization steps were usually reduced and the entire preparation proceeded more smoothly. The protein content of the D N A preparations can be estimated on the basis that a z6o mf~/z3o m# ratio of 2.2 corresponds to about o.3-o.5 °o protein 1°. The 26o m y 2 3 o m~t ratio is probably more significant than the often quoted 26o m/~/28o mi~ ratio, because the former reflects absorbance due to the amide group, present in all peptides, whereas the latter is sensitive only to the aromatic amino acids. Kinetic analysis of DNA-liberation from two cell lines (adenovirus-type 7 and h u m a n embryonic lung) are shown in Figs. I and 2. The results indicate t h a t m a n y hours or days of contact are necessary to obtain a high yield of DNA. These observations are consistent with the data plotted in Fig. 3, showing the yield of Biochim. Biophys. ~4cIa, ~38 (1967) 506 51z
'
i
ba
J
vo .q v
0o
%
t
DNA
20o 91o 12oo 270 65o 47 ° 4500 320 464 ° 80 420 2300 45 ° lO3O 850 12oo 250 186o
g cells
o/
5.6 25.0 33.0 18.8 45 5.2 50.0 3.6 51.o 2.6 13.5 74.0 12.6 29.0 > 5° 80.6 16.8 --
Theor.
o~ .o
extracted
ygDNAI
• P r e p a r a t i o n s u s e d in k i n e t i c s t u d i e s (see F i g s . I a n d 2).
i7 25- 3° 24 28 2 4 - 28 43 ° ; 7 17o 620 600 1 4 2 2 - 26 19o I 4 380 500 95 ]2o 12oo lOO
1 2 I i '2 i 2 3 i I 2 3 I 2 I I 2 I
Ha2°-I Ha2°-I H a 2 ° [aH~-I HaLiv-I HaLiv-I Ad7HaT II Ad7HaT-11 Ad7HaT-I[ Ad7HaT-Ill* ]?YTFHa[aHI-I PYTFHa[aHI-I P Y T F H a ~ 3 H J -I SPTFHa-I SPTFHa-1 HEL-I * ADI2TFHEL-I ADI2TFHEL-I 1,2B- [
(h)
Extraclion Contact with No. reagents
2"issue source
PHB, PHB, NSA 8HQ 8HQ 8HQ, 8HQ, 8HQ, 8HQ PHB PHB PHB PHB PHB, 8HQ 8HQ 8HQ 8HQ NSA
PHB PHB PHB
NSA NSA
Additional reagents z~sed
1.77 1.69 1.86 1.55 i .85 1.92 1.84 1.86 1.85 1.85 1-83 1.87 1.88 1.84 1.85 1.81 1.83 1.89
260/280
--2.11 1.24 1.91 2.66 2.35 2.36 2.36 -1.94 -1.78 2.30 2.44 2.25 2.20 2.36
260,/230
Absorbance ratios (mlt )
87.2 86. 7 86,0 87. 3 86.6 87.2 86.6 86.6 87. 4 _ 86.8 86.2 86. 3 86.6 87-2 84. 4 84.2 --
7"m ( ± o.8 ° )
36 28 39 19 3° 38 39 41 41 _ 35 36 38 4° 38 2>3 ° 39 --
°/o Hyperchromism
Thermal denaturation data
T h e f o l l o w i n g a b b r e v i a t i o n s for t h e cell l i n e s a n d t i s s u e s h a v e b e e n u s e d : H a , h a m s t e r ; H , h u m a n ; 2 °, s e c o n d a r y - c u l t u r e d f i b r o b l a s t s ; L i v , a d u l t l i v e r ; A d 7 a n d Ad12, a d e n o v i r u s - t y p e 7 a n d 12, r e s p e c t i v e l y ; P Y , p o l y o m a v i r u s ; T F , t r a n s f o r m e d ; T, t u m o r ; SP, s p o n t a n e o u s ; ( H ) E I . , ( h t m l a n ) e m b r y o n i c l u n g ; I¢B, h u m a n t,:B cells. All d e s i g n a t i o n s r e f e r t o c e l l s p r o p a g a t e d i n t i s s u e c u l t u r e e x c e p t for H a L i v ( a d u l t h a m s t e r l i v e r ) . T h e I { o m a n n u m e r a l r e f e r s to t h e c e l l b a t c h . To s o m e cell c u l t u r e s aH w a s a d d e d in t h e f o r m of I 3 l e - a H ~ t h y m i d i n e . A l l t h e extractions were carried out at room temperature using phenol, sodium trichloroacetate and sodium dodecvl sulfate. In addition some p r e p a r a t i o n s c o n t a i n e d o r g a n i c s a l t s a s i n d i c a t e d in t h e t a b l e : P H B , p - h y d r o x v b e n z o a t e ; N S A , 2 - n a p h t h a l e n e s u l f o n a t e ; 8 H Q , 8 - h y d r o x y q u i n o l i n e . T h e w e i g h t of t h e cells w a s e s t i m a t e d o n t h e b a s i s of t h e i r v o l u m e a f t e r c e n t r i f u g a t i o n a t 1 5 o o - 2 5 o o r e v . / m i n for i o 15 m i n , a s s u u l i n g a d e n s i t y of 1.1 g / m l . T h e y i e l d of D N A w a s b a s e d o n a n e x t i n c t i o n of 4 o / ~ g / A m l (1 c m p a t h - l e n g t h a t 26o m / , ) . T h e D N A c o n t e n t of t h e cells w a s d e t e r m i n e d b y t h e d i p h e n y l a m i n e r e a c t i o n of t3URTON 6 o r DXSCHE v. T h e m e l t i n g t e m p e r a t u r e (Tin) a n d % h y p e r c h r o m i s m w e r e d e t e r m i n e d in 1.o x SSC xs (no c o r r e c t i o n s w e r e m a d e for t h e r m a l e x p a n s i o n of t h e s o l u t i o n ) .
CHARACTERISTICS OF D N A PREPARATIONS
TABLE
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440
Fig. ~. A c t i o n of p h e n o l a n d o t h e r e x t r a c t i n g r e a g e n t s on t h e re l e a s e of D N A from h u m a n e n> b r y o n i c l u n g cells. To 2 g of t h e s e cells were a d d e d 15 ml o. 3 M s o d i u m t r i c h l o r o a c e t a t e (pH 7.2), I ~}{jin s o d i u m d o d e c y l s u l f a t e a n d 4 m l r e d i s t i l l e d phenol , o.i % in 8 - h y d r o x y q u i n o l i n e . O r d i n a t e is t h e c o n c e n t r a t i o n of D N A in t h e a q u e o u s phase. Th e r e a c t i o n w a s c a r r i e d o u t a t 23 26 °. E x p l a n a t i o n of a r r o w s : A (79 h), s o l u t i o n frozen for 24 h (time n o t i n c l u d e d on a bs c i s s a ); B (18~ h), o.25 vol. c h l o r o f o r n l - o c t a n o l (24 : i, v / v ) added. A t 226 h t h e a q u e o u s l a y e r w a s r e m o v e d a n d r e p l a c e d w i t h IO ml fresh s o d i u m t r i c h l o r o a c e t a t e - s o d i u m d o d e c y l sulfate. The c u r v e w a s d r a w n a c c o r d i n g to t h e e q u a t i o n g i v e n in t h e t e x t . I
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Fig. 2. A c t i o n of p h e n o l a n d e x t r a c t i n g r e a g e n t s on tile release of I ) N A from cells c u l t u r e d from a t u m o r i n d u c e d in a h a m s t e r by' a d e n o v i r u s - t y p e 7. To 1. 5 g of t he cells were a d d e d ~i. 5 ml o. 3 M s o d i u m t r i c h l o r o a c e t a t e (pH 7.2), I o~) in s o d i u m d o d e c y l s ul fa t e a nd i. 5 ml r e d i s t i l l e d phenol, o.I O,/o in 8-hvdroxvquinoline.. O r d i i m t e is t o t a l nag D N A re l e a s e d pe r g of cells. The r e a c t i o n was c a r r i e d o u t a t 23 26:. E x p l a n a t i o n of a r r o w s : A (248 h), o.43 vol. s odi ul n t ri c hl oroa c e t a t e s o d i u m d o d e c v l s u l f a t e a d d e d to soln.; B (297 h), a q u e o u s s o l u t i o n r e m o v e d a nd r e p l a c e d w i t h 7 ml s o d i u m t r i c i a l o r o a c e t a t e s o d i m n d o d e c v l s u l f a t e plus 2 ml phe nol ; C (315 h), a q u e o u s s o l u t i o n r e m o v e d aIxl c o m b i n e d w i t h the s o l u t i o n p r e v i o u s l y r e m o v e d a t 297 h; I) (345 h), o.25 vol. chloroforn: o c t a n o l ( 2 4 : t , v/v) added. Fig. 3. Yield of I)NA ol)tained from s e v e r a l t y p e s of tis~.ue as a f u n c t i o n of tile numl )e r of h o u r s t h e c e l l u l a r m a t e r i a l r e m a i n e d in c o n t a c t w i t h t h e e x t r a c t i n g r e a g e n t s . O, 1 lu 2"; ~ , H a l.iv; A, A d 7 H a t ; V , P Y T F H a ; O , S P T F Ha. The d a t a a n d a b b r ( v i a t i o n s for tile cell t y p e s are t a k e n from T a b l e 1. T o t a l I ) N A w a s d e t e r m i n e d l:y the d i p h e n y q a m i n e r e a c t i o n of I{URTON'i or I)ISCHIC7.
DNA from several cell types as a function of the hours of contact with the extracting reagents. The kinetics of deproteinization may be outlined by the following reaction steps: /"1 DNA-protein-~-R +~-~DNA protein R k,, Biochim. Biophys..tcta, 138 (1967) 506 512
(I)
STUDIES ON THE DEPROTEINIZATION OF DNA
511
k3
D N A - p r o t e i n - R ~ DNA +protein + R
(2)
k4 If
d[DNA-Pr-R] dt
= o (steady-state condition) and k4
o
(a reasonable
assumption because the bulk of the protein precipitates out of solution after separation from the DNA) one can apply the MICHAELIS-MENTEN1° equation:
d [DNA ] dt
k s ~R] --
i+
Km
(3)
[DN A-protein !
where [DNA ], [DNA-protein~ and [R l are the concentrations of DNA, DNA-protein and combined extracting reagents, respectively, and K m = (k2÷ka)/kl. Integrating, one obtains:
[-
~DNAIr
1
Km In ([DNA If-- [DNAJJ -- [DNA~ = ka[R]t
(4)
where [DNA ]r is the final concentration of DNA. For the deproteinization of human embryonic lung cell DNA I(m and ks have the values 34 °/,g/ml and 3.5" IO 6.h-1 respectively, where [DNAir is taken as 313/,g/ml and [RI ~ 800 mg/ml phenol. The good correspondence between the experimental points and Eqn. 4 as shown in Fig. I is consistent with the reaction steps outlined in Eqn. I and 2. It is noteworthy that the addition of various organic salts to the deproteinizing reagents, as shown in Table I and Fig. 3, did not change the general feature of the need for long periods of deproteinization in order to obtain maximum yields of DNA. There is a dearth of references to the kinetics of DNA extraction although several investigators have reported long-term DNA extractions. MIRSKY AND POLLISTER11 described a 48-h chloroform-octanol extraction at pH i o - i i for removal of DNA-bound protein (a condition which causes denaturation of DNA). DOUNCE AND MONTY12 reported that a 24-h stirring period in I.O M NaCI was needed before a satisfactory yield of calf-thymus DNA could be obtained by further treatment with sodium dodecyl sulfate, a result consistent with reference by CHARGAFF13 to a 4-day stirring period of calf-thymus nucleoprotein in saturated NaC1. The limitation of the solubility of the nucleoprotein as a factor in DNA release has been discussed by COLTER, BROWN" AND ELLEM 14, who employed phenol to extract DNA from mouse Ehrlieh aseites cells, mouse liver, and pneumocoecus. It is clear that better yields of DNA from the tissue types studied here can be obtained by allowing the DNA-extraeting reagents to remain in contact with the cells or tissue. How general this feature is will depend on the outcome of studies with other tissues. Optimum yields of DNA are important not only in terms of economy but because the possibility remains that the composition of extracted DNA may not faithfully represent the DNA in vivo if all or nearly all of the DNA is not extracted. Biases in the DNA composition could result if the procedure favored extraction of DNA with either a higher or lower G + C content, or DNA that is bound to certain proteins and not to others, e.g., repressed DNA as opposed to derepressed DNA. Such a consideration is important in view of the fact that a small Biochim. Biophys. Acta, 138 (1967) 5o6-512
512
17,. S. YOLLES, G. FREEMAN
f r a c t i o n of t h e D N A i n a d i f f e r e n t i a t e d cell is i n a d e r e p r e s s e d s t a t e a t a n y g i v e n t i m e 15. I t is l i k e l y t h a t t h e i n f l u e n c e of t i m e o n D N A d e p r o t e i n i z a t i o n h a s n o t b e e n a d e q u a t e l y s t u d i e d . Y e t t h e c h e m i s t r y of d e p r o t e i n i z a t i o n is a k e y t o u n d e r s t a n d i n g t h e n a t u r e of b o n d s i n t h e n u c l e o p r o t e i n c o m p l e x w h i c h , if f u l l y e l u c i d a t e d , c o u l d p r o v i d e i n f o r m a t i o n b a s i c t o t h e u n d e r s t a n d i n g of g e n e r e p r e s s i o n .
ACKNOWLEDGEMENTS
W e t h a n k D r . I. V. SULTANIAN for s u p p l y i n g t h e t i s s u e c u l t u r e cells a n d for h e l p f u l d i s c u s s i o n s . W e a r e p a r t i c u l a r l y i n d e b t e d t o Mr. LAWRENCE HOOSER, Mr. RICHARD J . JONES, a n d Mrs. AUDREY KUEHN for t h e i r e x c e l l e n t t e c h n i c a l a s s i s t a n c e a n d t o D r . F.-C. CHAO a n d Mrs. ANN GRIFFIN for s u p p l y i n g d a t a c o n c e r n i n g t h e t o t a l D N A c o n t e n t of s e v e r a l of t h e h a m s t e r cell lines. T h i s w o r k w a s s u p p o r t e d i n p a r t b y N a t i o n a l I n s t i t u t e s of H e a l t h G r a n t x S O I F R - o 5 5 2 2 f r o m t h e F a c i l i t i e s R e s o u r c e s B r a n c h a n d C o n t r a c t No. P H 4 3 - 6 5 - 4 3 , CCNSC, N C I .
]~EFERENCES I K. S. ]'~IRBY, JProgr. Exptl. Tumor Res., 2 (I96I) 29i. 2 1,2. S. 1,1IRBY, in J. N. DAVIDSON AND \V. g. COHN, Progress in NT~cleic Acid Research and Molecular Biology, Vol. 3, Academic Press, New York, 1964, p. I. 3 K. S. KIRBY, Biochenz. J., 66 (1957) 4954 J- MARMUR, in S. P. COLOWlCK AND N. O. NAPLAX, Methods in ]Snzyuzology, Vol. V[, Acadenlic Press, New York, 1963, p. 726. 5 P. M. B. -WALKER AND A. MCLAREN, .]. 3Iol. Biol., 12 (1965) 3946 K. BURTON, Biochem. J., 62 (1956 ) 315 . 7 Z. I)ISCHE, in E. CHARGAFF AND J. N. DAVII)SON, The Nucleic Acids, Vol. i, Academic Press, New York, I955, p. 285. 8 I. V. SULTANIAN AND G. lgREEMAN, Science, 154 (1966) 665. 9 L. ~{ICHAELIS AND M. L. MENTEN, Bioche~n. Z., 49 (1913) 333. 10 J. MARMUR, J. Mol. Biol., 3 (1961) 208. 11 A. E. MIRSKY AND A. "~\r. POLLISTER, ,]. Gen. Physiol., 3 ° (1946) 117. 12 A. L. 1)OUNCE AND K. J. MONTY, J. Biophys. Biochem. Cytol., 1 (J955) 15513 E. CHARC,AI~F, in E. CHARGAVF ANt) J. N. DaV]DSON, The Nucleic .tcids, Vol. I, Academic Press, New York, 1955, p. 3o7 . J4 J. S. CCmTER, 1¢. A. BROWN AND 1~. A. O. ELLEM, Biochim. Biophys...Icta, 55 (1962) 31. ]5 J. PAUL AND N. S. GILMOUR, .]. Mol. Biol., 16 (z966) 242. 16 J. MARMUR AND l ), DOTY, .[. Mol. Biol., 5 (]962) lO9. Biochim. Biophys. ~4cta, I3S (1967) 506 512