- Email: [email protected]
Isolation and characterization of DNA from fixed cells and tissues
0 1968 by Academic Press Inc. Experimental
47
Cell Research 50, 47-53 (196s)
ISOLATION
AND CHARACTERIZATION
FROM FIXED FRANCES E. ARRIGHI,
OF DNA
CELLS AND TISSUES’
JANET BERGENDAHL
and MANLEY
MANDEL
Sections of Cytology and Molecular Biology, Department of Biology, The University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77025, USA Received
June 1, 1967
and frozen tissues of higher organisms have been used for extracting deoxyribonucleic acid (DNA) by conventional methods [S]. However, such procedures are not always convenient or practicable. It is sometimes not desirable to store tissue samples in a freezer, especially when such facilities are not available, such as on field trips. The present communication describes a method for extracting DNA from tissues and cells fixed with a variety of common fixatives. Biophysical data are presented to compare the properties of DNA isolated from fixed material with that isolated from fresh tissues by the conventional methods.
FRESH
MATERIALS
AND
METHODS
In situ tissues.-Liver samples of the Chinese hamster, Cricetulus griseus, were used. They were either used directly for DNA extraction or were fixed and the DNA extracted later. In vitro cells.-Cells of the Chinese hamster clone Don C were used extensively for this study. A few DNA extractions were made from the aneuploid mouse cell strain LM. Bacteria.-The only bacteria fixed were Escherichia coli, strain K 12. Fixatives.-The following fixatives were used: 95 per cent ethanol; abs. methanol; 1 abs. methanol: 9 ethanol; 5 abs. methanol: 5 ethanol; 3 methanol: I glacial acetic acid; 2-propanol; acetone; and IO per cent formalin (prepared in a phosphate buffer). Fixation times varied from 4 to 66 days. Procedure.-Since it was necessary to modify the Marmur [6] procedure, a brief description of our procedure follows. (I) Remove cells from culture flask with 0.1 per cent Pronase solution. Collect cell pellet by centrifugation and disperse cells in the fixative. Freeze control cells in an equal volume of medium. (2) After the fixation period wash the cells three times with 70 per cent ethanol 1 Supported in part by Research Grants DRG-269 from Damon Runyon Memorial Fund for Cancer Research
and GB-1867 from National
Science Foundation. Experimental
Cell Research 50
Frances E. Arrighi,
Janet Bergendahl
and ibfanley Mandel
and twice with saline EDTA (0.15 M NaCl, 0.10 M EDTA, pH 8.0). Acetone fixed cells were washed twice with absolute ethanol prior to the 70 per cent ethanol washing. (3) Collect cells in saline EDTA, add Pronase (100 ,ug/ml), and sodium dodecyl sulfate to a final concentration of 0.25 per cent. Adjust the pH in formalin fixed samples to 7.6-8.0, and incubate the mixture at 37°C until lysis occurs. Breakdown of fixed cell membranes may take longer than 24 h. (4) Add an equal volume of phenol saturated with saline EDTA and adjust pH to 7.0. The saturated phenol may be replaced with sodium perchlorate at a final concentration of 1.0 M. If sodium perchlorate is used the mixture is shaken at 37°C for 60 min, then CHCl,-isoamyl alcohol (24:l) (Sevag) is added. (5) Shake, centrifuge, and carefully remove the supernatant containing the DNA. (6) Ethanol precipitate the DNA, spool, and redissolve in dilute saline-citrate solution (l/IO SSC, see [6]). (7) Adjust to standard saline citrate concentration (SSC) and treat with RNase (50 pg/ml) at 37°C for 30 min. (8) Treat with Pronase (100 pg/ml) at 37°C for 60 min. (9) Add equal volume of CHCl,-isoamyl alcohol (24:l) and shake for 15 min. Centrifuge and carefully remove the supernatant. (10) Precipitate with ethanol, spool, and dissolve in l/IO SSC. (11) Isopropanol precipitate as described by Marmur [6]. (12) Dissolve the glassy precipitate in l/IO SSC. When dissolved adjust concentration to standard SSC. The procedure for isolating DNA from liver tissue and bacteria was identical except for the following: segments of liver were removed immediately upon sacrifice and minced with a scalpel in the respective fixatives. The fragments and excess fixative were placed in tightly stoppered bottles for various periods of time. All samples were stored at room temperature except the acetone fixed samples which were stored at 4°C. Melting temperature determinations were done according to Mandel and Marmur [4]. The CsCl buoyant density determinations were obtained in a Spinco Model E analytical ultracentrifuge as described by Mandel ef al. [5]. Reference DNA of bacteriophage SP8 (1.742 g/cm3) and Caulobacfer sp. CB2 (1.726 g/cm”) were both included in each gradient. RESULTS Isolation of DNA was not difficult from materials treated with most fixatives except methanol and formalin. If sodium perchlorate at a final concentration of 1 .O M was used to replace phenol in the first deproteinization, DNA could be isolated from methanol fixed cells. Obtaining DNA from formalin fixed tissue culture cells was not always successful, and no DNA was obtained from minced tissues after formalin treatment. m7hen either phenol or sodium perchlorate was used for the first deproteinization after formalin fixation, no DNA could be recovered in the supernatant. However, DNA could be recovered from the interface of the first deproteinization if sodium perchloExperimental
Cell Research 50
Isolation
and characterization
of DNA
rate was used. This interface was redissolved in dilute salt solution (l/10 SSC) with no alteration in the rest of the procedure. The purity of DNA prepared from fixed mammalian material compares favorably with DNA obtained from fresh or frozen samples, except after formalin fixation; although one sample from liver after methanol fixation had a relatively low absorbance ratio (260 mp/230 mp). All samples of bacterial DNA after fixation had lower ratios than normally obtained. Table 1 gives the spectral absorption ratios at 26Oj230 and 2601280 m,u. It appears TABLE
1. Absorption spectral ratios of all samples of DNA isolated from various species after treatment with various fixatives Control
samples were not fixed.
Specimen
Treatment
Cricetulus griseus, Don C cells (Chinese hamster)
Unfixed MEOH MEOH 5 MEOH: ETOH 1 MEOH:Q ETOH ETOH 3 MEOH:l HAC Acetone 2-Propanol 10 y0 formalin 10 y0 formalin 10 y0 formalin 10 y0 formalin 10 % formalin
Cricefulus
Unfixed 2-Propanol 2-Propanol ETOH MEOH 1 MEOH:Q
griseus (liver
tissue)
ETOH
Mouse LM cells
Unfixed 2-Propanol
Escherichia
Unfixed 10 y0 formalin 2-Propanol MEOH 1 MEOH:Q ETOH
4 - 681806
coli, K12
Time (days)
260 m,u/ 230 m,u
260 m,u/ 280 m,u
0 11 56 66 11 11 11 21 11 6 8 9 11 18
2.14 1.74 2.12 2.20 2.23 2.18 2.19 2.16 2.27 2.21 1.68 1.75 1.64 1.77
1.88 1.84 1.94 1.87 1.88 1.87 1.84 1.86 1.86 1.88 1.83 1.84 1.84 1.82
0 14 39 24 14 14
2.51 2.02 1.96 2.04 1.56 1.96
1.88 1.91 1.91 1.86 1.85 1.87
0 23
2.45 2.27
1.89 1.86
0 4 4 4 4
2.11 0.48 1.39 1.28 1.39
1.84 1.42 1.79 1.78 1.80
Experimental
Cell Research 50
Frances l?. Arrighi,
50
Janet Bergendahl
and Manley
Mandel
that the longer the formalin treatment, the lower the ratio. In all cases contaminating material appeared to affect the absorbance at 230 m,u more markedly than at 280 m,u. The C&l buoyant density values are given in Tables 2 and 3. Table 3 presents the values obtained from the tissue culture cell strain Don C, and TABLE 2. Characferistics of DNA isolated from the in vitro cell strain Don C of the Chinese hamster with and without treatment of various fixatives for various intervals of time
Treatment
Time (days)
Unfixed ETOH MEOH MEOH 5 MEOH: 5 ETOH 1 MEOH:Q ETOH 3 MEOH:l HAC 2-Propanol Acetone 10 y0 formalin 10 y0 formalin 10 % formalin 10 y0 formalin 10 y0 formalin
11 11 56 66 11 11 11 21 6 8 9 11 18
0
Mean
Buoyant density (g/cm3 CsCl) 1.700 1.698 1.699 1.700 1.700 1.700 1.701 1.699 1.701 1.696 1.692 1.693 1.696 1.688
% HyperTnl
chromicity
Compositional heterogeneity
86.1 86.5 86.6 -a -
40 41 39 -
5.86 5.37 5.86 -
86.4 86.6 87.2 86.5
44 42 45 40 -
6.34 4.88 6.59 5.32 -
86.2
42 -
7.32 -
86.0 -
40 -
7.32 -
86.5) 0.4
41.3_+2
6.110.9
a Not examined.
Table 3, those from other materials. Again, the values are the same as those of the control sample except after formalin fixation. These values varied from sample to sample, but were always lower than those of fresh or frozen material. This is true for both mammalian cells and bacteria. In general the longer the fixation treatment, the lower the value. One DNA sample isolated after formalin fixation was determined a second time in CsCl, and the value coincided with the first determination. These values compare well with those obtained by other investigators for E. coli and rodents [l, 9, 111. The satellite or minor band of mouse cells did not appear Experimental
Cell Research 50
Isolation
and characterization
of DNA
3. Cesium chloride buoyant density values of DNA isolated from the bacterium, Escherichia coli, strain K12, livers of Cricetulus griseus (Chinese hamster), and the in vitro mouse cell strain LM after treatment with various fixatives for various periods of time. TARI.E
Specimen Escherichia
Cricefulus
coli
griseus (liver
Unfixed 10 %formalin 2-Propanol MEOH 1 MEOH:O ETOH tissue)
Mouse LM cells
a Satellite
Time (days)
Treatment
Unfixed 2-Propanol 2-Propanol ETOH MEOH 1 MEOH:Q Unfixed 2-Propanol
ETOH
Buoyant density (g/cm3 CsCI)
0 4 4 4 4
1.710 1.703 1.711 1.711 1.711
0 14 39 24 14 14
1.699 1.700 1.700 1.700 1.700 1.701
0 23
1.699 1.699
85
I II)
1.689a 1.692’
band.
Fig. I.-Melting temperatures of three samples of DNA isolated from the in vitro cell strain Don C. Circles, Nontreated cells; stars, cells treated with formalin; squares, cells treated with 3 parts methanol: 1 part acetic acid. The T, values are 86.1, 86.2, and 86.6, respectively. Abscissa: Temperature OC; ordinate: relative absorbance.
Experimenfal
16
Cell Research 50
52
Frances E. Arrighi,
Janet Bergendahl
and Manley
Mandel
to be significantly altered in amount or position in the gradient when DNA from 2-propanol fixed cells was examined. The CsCl buoyant density values from six Chinese hamster DNA samples not included in any of the tables gave a mean value of 1.700 g/cm3 with a standard deviation of +O.OOlZ. These samples of DNA were isolated from both male and female in vitro cell lines and fresh liver. Further statistical analysis of CsCl buoyant density values of 44 runs in the Model E ultracentrifuge of DNA of Caulabacter CR 2 gave a mean value of 1.7255 with a standard deviation of +_O.OOOS [3]. Thus the low values obtained after formalin fixation are significant deviations from the mean. Contrary to the differences seen in buoyant densities, melting curves showed no alteration between the DNA from control samples and that from cells fixed in any of the fixatives. Tables 2 and 3 give the T,,, values and per cent hypochromicity, and Fig. 1 shows three representative melting curves. The T,,, values compare favorably with those of mammalian DNA reported by other investigators [ 7 1. Table 2 also presents the compositional heterogeneity [4, lo] for some samples. These gross parameters are essentially identical for all samples.
DISCUSSION
From the data just presented it appears that mammalian DNA can be extracted satisfactorily from fixed cells or tissues. The cause of the lower CsCl buoyant density values after formalin fixation is unknown. It has been shown that formaldehyde crosslinks fibrous proteins [S] as well as soluble proteins [2]. It is possible that some type of linkage also occurred during fixation which altered the macromolecules in such a way that enzymatic purification of DNA was not efficient. This phenomenon is being further investigated. Of all the fixatives used, 2-propanol emerges as the most effective and convenient, but there are advantages to the use of ethanol on field trips unless the materials are to be mailed. SUMMARY
DNA was isolated from cultured mammalian cells, liver tissue and Escherichia coli after treatment with the common laboratory fixatives for various periods of time. The cultured cells included the mouse cell strain LM and the Chinese hamster cell strain Don C. Liver tissue was obtained from young Experimental
Cell Research 50
Isolation
and characterization
of DXA
53
adult Chinese hamsters. Ultraviolet spectral absorbance ratios, CsCl buoyant melting curves and hyperchromicity were used to density determinations, characterize the DNA samples. DNA was excellent with most fixatives, but not Recovery of “normal” when formalin was used. Formalin fixation interfered with the isolation procedure, and when DNA was extracted the buoyant densities in CsCl were significantly different from the DNA extracted from unfixed materials, but the T,, values were not different. The gift of an analytical ultracentrifuge and accessories by the Mr and Mrs T. N. Law Foundation is gratefully acknowledged. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
ANTONOV, A. S., Usp Sovrem Biol. 60, 161 (1965). FRAENKEL-CONRAT, H. and OLCOTT, H. S., J. Am. Chem. Sac. 70, 2673 (1948). MANDEL, M., J. Gen. Microbial. 43, 273 (1966). MANDEL, M. and MARMUR, J., in L. GROSSMAN and K. MOLDAVE (eds), Methods in Enzymology, vol. 12. Academic Press, New York. In press. MANDEL, M., SCHILDKRAUT, C. L. and MARMUR, J., in L. GROSSMAN and K. MOLDAVE (eds), Methods in Enzymology, vol. 12. Academic Press, New York. In press. MARMUR, J., J. Mol. Biol. 3, 208 (1961). MARMUR, J. and DOTY, P., J. Mol. Biol. 5, 109 (1962). MASON, P. and GRIFFITH, J. C., Nature 203, 484’(1964). SCHILDKRAUT, C. L., MARMUR, J. and DOTY, P., J. Mol. Biol. 4, 430 (1962). SUEOKA, N., J. Mol. Biol. 3, 31 (1961). SZYBALSKI, W., in L. GROSSMAN and K. MOLDAVE (eds), Methods in Enzymology, vol. 12. Academic Press, New York. In press.
Experimental
Cell Research 50
47
Cell Research 50, 47-53 (196s)
ISOLATION
AND CHARACTERIZATION
FROM FIXED FRANCES E. ARRIGHI,
OF DNA
CELLS AND TISSUES’
JANET BERGENDAHL
and MANLEY
MANDEL
Sections of Cytology and Molecular Biology, Department of Biology, The University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77025, USA Received
June 1, 1967
and frozen tissues of higher organisms have been used for extracting deoxyribonucleic acid (DNA) by conventional methods [S]. However, such procedures are not always convenient or practicable. It is sometimes not desirable to store tissue samples in a freezer, especially when such facilities are not available, such as on field trips. The present communication describes a method for extracting DNA from tissues and cells fixed with a variety of common fixatives. Biophysical data are presented to compare the properties of DNA isolated from fixed material with that isolated from fresh tissues by the conventional methods.
FRESH
MATERIALS
AND
METHODS
In situ tissues.-Liver samples of the Chinese hamster, Cricetulus griseus, were used. They were either used directly for DNA extraction or were fixed and the DNA extracted later. In vitro cells.-Cells of the Chinese hamster clone Don C were used extensively for this study. A few DNA extractions were made from the aneuploid mouse cell strain LM. Bacteria.-The only bacteria fixed were Escherichia coli, strain K 12. Fixatives.-The following fixatives were used: 95 per cent ethanol; abs. methanol; 1 abs. methanol: 9 ethanol; 5 abs. methanol: 5 ethanol; 3 methanol: I glacial acetic acid; 2-propanol; acetone; and IO per cent formalin (prepared in a phosphate buffer). Fixation times varied from 4 to 66 days. Procedure.-Since it was necessary to modify the Marmur [6] procedure, a brief description of our procedure follows. (I) Remove cells from culture flask with 0.1 per cent Pronase solution. Collect cell pellet by centrifugation and disperse cells in the fixative. Freeze control cells in an equal volume of medium. (2) After the fixation period wash the cells three times with 70 per cent ethanol 1 Supported in part by Research Grants DRG-269 from Damon Runyon Memorial Fund for Cancer Research
and GB-1867 from National
Science Foundation. Experimental
Cell Research 50
Frances E. Arrighi,
Janet Bergendahl
and ibfanley Mandel
and twice with saline EDTA (0.15 M NaCl, 0.10 M EDTA, pH 8.0). Acetone fixed cells were washed twice with absolute ethanol prior to the 70 per cent ethanol washing. (3) Collect cells in saline EDTA, add Pronase (100 ,ug/ml), and sodium dodecyl sulfate to a final concentration of 0.25 per cent. Adjust the pH in formalin fixed samples to 7.6-8.0, and incubate the mixture at 37°C until lysis occurs. Breakdown of fixed cell membranes may take longer than 24 h. (4) Add an equal volume of phenol saturated with saline EDTA and adjust pH to 7.0. The saturated phenol may be replaced with sodium perchlorate at a final concentration of 1.0 M. If sodium perchlorate is used the mixture is shaken at 37°C for 60 min, then CHCl,-isoamyl alcohol (24:l) (Sevag) is added. (5) Shake, centrifuge, and carefully remove the supernatant containing the DNA. (6) Ethanol precipitate the DNA, spool, and redissolve in dilute saline-citrate solution (l/IO SSC, see [6]). (7) Adjust to standard saline citrate concentration (SSC) and treat with RNase (50 pg/ml) at 37°C for 30 min. (8) Treat with Pronase (100 pg/ml) at 37°C for 60 min. (9) Add equal volume of CHCl,-isoamyl alcohol (24:l) and shake for 15 min. Centrifuge and carefully remove the supernatant. (10) Precipitate with ethanol, spool, and dissolve in l/IO SSC. (11) Isopropanol precipitate as described by Marmur [6]. (12) Dissolve the glassy precipitate in l/IO SSC. When dissolved adjust concentration to standard SSC. The procedure for isolating DNA from liver tissue and bacteria was identical except for the following: segments of liver were removed immediately upon sacrifice and minced with a scalpel in the respective fixatives. The fragments and excess fixative were placed in tightly stoppered bottles for various periods of time. All samples were stored at room temperature except the acetone fixed samples which were stored at 4°C. Melting temperature determinations were done according to Mandel and Marmur [4]. The CsCl buoyant density determinations were obtained in a Spinco Model E analytical ultracentrifuge as described by Mandel ef al. [5]. Reference DNA of bacteriophage SP8 (1.742 g/cm3) and Caulobacfer sp. CB2 (1.726 g/cm”) were both included in each gradient. RESULTS Isolation of DNA was not difficult from materials treated with most fixatives except methanol and formalin. If sodium perchlorate at a final concentration of 1 .O M was used to replace phenol in the first deproteinization, DNA could be isolated from methanol fixed cells. Obtaining DNA from formalin fixed tissue culture cells was not always successful, and no DNA was obtained from minced tissues after formalin treatment. m7hen either phenol or sodium perchlorate was used for the first deproteinization after formalin fixation, no DNA could be recovered in the supernatant. However, DNA could be recovered from the interface of the first deproteinization if sodium perchloExperimental
Cell Research 50
Isolation
and characterization
of DNA
rate was used. This interface was redissolved in dilute salt solution (l/10 SSC) with no alteration in the rest of the procedure. The purity of DNA prepared from fixed mammalian material compares favorably with DNA obtained from fresh or frozen samples, except after formalin fixation; although one sample from liver after methanol fixation had a relatively low absorbance ratio (260 mp/230 mp). All samples of bacterial DNA after fixation had lower ratios than normally obtained. Table 1 gives the spectral absorption ratios at 26Oj230 and 2601280 m,u. It appears TABLE
1. Absorption spectral ratios of all samples of DNA isolated from various species after treatment with various fixatives Control
samples were not fixed.
Specimen
Treatment
Cricetulus griseus, Don C cells (Chinese hamster)
Unfixed MEOH MEOH 5 MEOH: ETOH 1 MEOH:Q ETOH ETOH 3 MEOH:l HAC Acetone 2-Propanol 10 y0 formalin 10 y0 formalin 10 y0 formalin 10 y0 formalin 10 % formalin
Cricefulus
Unfixed 2-Propanol 2-Propanol ETOH MEOH 1 MEOH:Q
griseus (liver
tissue)
ETOH
Mouse LM cells
Unfixed 2-Propanol
Escherichia
Unfixed 10 y0 formalin 2-Propanol MEOH 1 MEOH:Q ETOH
4 - 681806
coli, K12
Time (days)
260 m,u/ 230 m,u
260 m,u/ 280 m,u
0 11 56 66 11 11 11 21 11 6 8 9 11 18
2.14 1.74 2.12 2.20 2.23 2.18 2.19 2.16 2.27 2.21 1.68 1.75 1.64 1.77
1.88 1.84 1.94 1.87 1.88 1.87 1.84 1.86 1.86 1.88 1.83 1.84 1.84 1.82
0 14 39 24 14 14
2.51 2.02 1.96 2.04 1.56 1.96
1.88 1.91 1.91 1.86 1.85 1.87
0 23
2.45 2.27
1.89 1.86
0 4 4 4 4
2.11 0.48 1.39 1.28 1.39
1.84 1.42 1.79 1.78 1.80
Experimental
Cell Research 50
Frances l?. Arrighi,
50
Janet Bergendahl
and Manley
Mandel
that the longer the formalin treatment, the lower the ratio. In all cases contaminating material appeared to affect the absorbance at 230 m,u more markedly than at 280 m,u. The C&l buoyant density values are given in Tables 2 and 3. Table 3 presents the values obtained from the tissue culture cell strain Don C, and TABLE 2. Characferistics of DNA isolated from the in vitro cell strain Don C of the Chinese hamster with and without treatment of various fixatives for various intervals of time
Treatment
Time (days)
Unfixed ETOH MEOH MEOH 5 MEOH: 5 ETOH 1 MEOH:Q ETOH 3 MEOH:l HAC 2-Propanol Acetone 10 y0 formalin 10 y0 formalin 10 % formalin 10 y0 formalin 10 y0 formalin
11 11 56 66 11 11 11 21 6 8 9 11 18
0
Mean
Buoyant density (g/cm3 CsCl) 1.700 1.698 1.699 1.700 1.700 1.700 1.701 1.699 1.701 1.696 1.692 1.693 1.696 1.688
% HyperTnl
chromicity
Compositional heterogeneity
86.1 86.5 86.6 -a -
40 41 39 -
5.86 5.37 5.86 -
86.4 86.6 87.2 86.5
44 42 45 40 -
6.34 4.88 6.59 5.32 -
86.2
42 -
7.32 -
86.0 -
40 -
7.32 -
86.5) 0.4
41.3_+2
6.110.9
a Not examined.
Table 3, those from other materials. Again, the values are the same as those of the control sample except after formalin fixation. These values varied from sample to sample, but were always lower than those of fresh or frozen material. This is true for both mammalian cells and bacteria. In general the longer the fixation treatment, the lower the value. One DNA sample isolated after formalin fixation was determined a second time in CsCl, and the value coincided with the first determination. These values compare well with those obtained by other investigators for E. coli and rodents [l, 9, 111. The satellite or minor band of mouse cells did not appear Experimental
Cell Research 50
Isolation
and characterization
of DNA
3. Cesium chloride buoyant density values of DNA isolated from the bacterium, Escherichia coli, strain K12, livers of Cricetulus griseus (Chinese hamster), and the in vitro mouse cell strain LM after treatment with various fixatives for various periods of time. TARI.E
Specimen Escherichia
Cricefulus
coli
griseus (liver
Unfixed 10 %formalin 2-Propanol MEOH 1 MEOH:O ETOH tissue)
Mouse LM cells
a Satellite
Time (days)
Treatment
Unfixed 2-Propanol 2-Propanol ETOH MEOH 1 MEOH:Q Unfixed 2-Propanol
ETOH
Buoyant density (g/cm3 CsCI)
0 4 4 4 4
1.710 1.703 1.711 1.711 1.711
0 14 39 24 14 14
1.699 1.700 1.700 1.700 1.700 1.701
0 23
1.699 1.699
85
I II)
1.689a 1.692’
band.
Fig. I.-Melting temperatures of three samples of DNA isolated from the in vitro cell strain Don C. Circles, Nontreated cells; stars, cells treated with formalin; squares, cells treated with 3 parts methanol: 1 part acetic acid. The T, values are 86.1, 86.2, and 86.6, respectively. Abscissa: Temperature OC; ordinate: relative absorbance.
Experimenfal
16
Cell Research 50
52
Frances E. Arrighi,
Janet Bergendahl
and Manley
Mandel
to be significantly altered in amount or position in the gradient when DNA from 2-propanol fixed cells was examined. The CsCl buoyant density values from six Chinese hamster DNA samples not included in any of the tables gave a mean value of 1.700 g/cm3 with a standard deviation of +O.OOlZ. These samples of DNA were isolated from both male and female in vitro cell lines and fresh liver. Further statistical analysis of CsCl buoyant density values of 44 runs in the Model E ultracentrifuge of DNA of Caulabacter CR 2 gave a mean value of 1.7255 with a standard deviation of +_O.OOOS [3]. Thus the low values obtained after formalin fixation are significant deviations from the mean. Contrary to the differences seen in buoyant densities, melting curves showed no alteration between the DNA from control samples and that from cells fixed in any of the fixatives. Tables 2 and 3 give the T,,, values and per cent hypochromicity, and Fig. 1 shows three representative melting curves. The T,,, values compare favorably with those of mammalian DNA reported by other investigators [ 7 1. Table 2 also presents the compositional heterogeneity [4, lo] for some samples. These gross parameters are essentially identical for all samples.
DISCUSSION
From the data just presented it appears that mammalian DNA can be extracted satisfactorily from fixed cells or tissues. The cause of the lower CsCl buoyant density values after formalin fixation is unknown. It has been shown that formaldehyde crosslinks fibrous proteins [S] as well as soluble proteins [2]. It is possible that some type of linkage also occurred during fixation which altered the macromolecules in such a way that enzymatic purification of DNA was not efficient. This phenomenon is being further investigated. Of all the fixatives used, 2-propanol emerges as the most effective and convenient, but there are advantages to the use of ethanol on field trips unless the materials are to be mailed. SUMMARY
DNA was isolated from cultured mammalian cells, liver tissue and Escherichia coli after treatment with the common laboratory fixatives for various periods of time. The cultured cells included the mouse cell strain LM and the Chinese hamster cell strain Don C. Liver tissue was obtained from young Experimental
Cell Research 50
Isolation
and characterization
of DXA
53
adult Chinese hamsters. Ultraviolet spectral absorbance ratios, CsCl buoyant melting curves and hyperchromicity were used to density determinations, characterize the DNA samples. DNA was excellent with most fixatives, but not Recovery of “normal” when formalin was used. Formalin fixation interfered with the isolation procedure, and when DNA was extracted the buoyant densities in CsCl were significantly different from the DNA extracted from unfixed materials, but the T,, values were not different. The gift of an analytical ultracentrifuge and accessories by the Mr and Mrs T. N. Law Foundation is gratefully acknowledged. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
ANTONOV, A. S., Usp Sovrem Biol. 60, 161 (1965). FRAENKEL-CONRAT, H. and OLCOTT, H. S., J. Am. Chem. Sac. 70, 2673 (1948). MANDEL, M., J. Gen. Microbial. 43, 273 (1966). MANDEL, M. and MARMUR, J., in L. GROSSMAN and K. MOLDAVE (eds), Methods in Enzymology, vol. 12. Academic Press, New York. In press. MANDEL, M., SCHILDKRAUT, C. L. and MARMUR, J., in L. GROSSMAN and K. MOLDAVE (eds), Methods in Enzymology, vol. 12. Academic Press, New York. In press. MARMUR, J., J. Mol. Biol. 3, 208 (1961). MARMUR, J. and DOTY, P., J. Mol. Biol. 5, 109 (1962). MASON, P. and GRIFFITH, J. C., Nature 203, 484’(1964). SCHILDKRAUT, C. L., MARMUR, J. and DOTY, P., J. Mol. Biol. 4, 430 (1962). SUEOKA, N., J. Mol. Biol. 3, 31 (1961). SZYBALSKI, W., in L. GROSSMAN and K. MOLDAVE (eds), Methods in Enzymology, vol. 12. Academic Press, New York. In press.
Experimental
Cell Research 50