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Rapid filter method for the microfluorometric analysis of DNA
ANALYTICAL
BIOCHEMISTRY
Rapid
Filter
69, 572-582
Method Analysis
(1975)
for the Microfluorometric of DNA’
ROSE ANN CATTOLICO~ AND SARAH P. GIBBS Department Montreal,
of Biology, McGill University, Quebec, Canada H3C 3Gl
Received June 10, 1975; accepted July 22, 1975 A rapid filter method for the microfluorometric analysis of DNA is reported in this communication. Cells collected on cellulose filters are subject to an assay sequence developed from a fluorometric method initially described by J. M. Kissane and E. Robins ((1958) J. Biol. Chem. 233, 184-188) for DNA quantitation. The assay is one of high specificity requiring no separation of DNA from other major cellular components. Samples containing as little as 0.2-0.3 pg of DNA have been analyzed by this filter method with a coefficient of variation for replicate standard samples of 3.3%. The DNA content of a number of cell types having different physiological characteristics has been determined by the use of this filter technique. The data obtained for Chlorella, Chlamydomonas, Ochromonas, and Stronglyocentrotus eggs were well within the values reported for these cells by other investigators using classical macromethods. We have taken advantage of the high specificity, sensitivity, and accuracy of this filter assay to determine DNA content during the synchronous growth cycle of the wall-less alga Olisthodiscus luteus using a single small volume culture as a sample source.
In 1958 Kissane and Robins (1) described a microfluorometric assay for the measurement of DNA. This assay requires the collection of microquantities of cells, a wash sequence to remove extraneous fluorescent materials, and finally an incubation at 60°C with 3,5-diaminobenzoic acid (DABA) dissolved in 4 N HCl. During the incubation step, the deoxyribose sugars released by the hydrolysis of the purine deoxyribonucleotides react in a highly specific manner with DABA to give a fluorescent product. The quantitation of DNA by this fluorometric assay has a number of technical advantages. Difficulties of nucleic acid degradation or incomplete recovery often encountered (see 2 and 3 for review) by the application of more classical DNA extraction methods (4-6) can be entirely avoided. Moreover, due to the high specificity of the fluorometric response, the presence of polysaccharides, protein, RNA, arabinose, and other substances which have been shown (2,7-9) i Supported by the National Research Council of Canada (Grant A-2921) and the Quebec Department of Education. * Present address: Department of Botany, University of Washington, Seattle, Washington 98195. 572 Copyri,ght All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
DNA
MICROFLUOROMETRIC
FILTER
ASSAY
573
to seriously affect the calorimetric and/or optical density measurement of DNA no longer presents a problem. It is interesting to note that although this fluorometric method has many obvious advantages, it has been rarely chosen by investigators. The tedious nature of microsample collection and manipulation has no doubt restricted its use. In this communication we wish to report upon a rapid method whereby the entire fluorometric assay sequence described above takes place on cellulose filters. The use of filters in the fluorometric determination of DNA would eliminate the need for precision in the pipetting of microquantities of cells (10) and wash materials (1). Cell adhesion to test tube surfaces during initial sample collection or during sample resuspension (11) would be eliminated. The tedious removal of microquantities of wash materials ( 1,lO) and therefore the possibility of sample loss during this often repeated step would be entirely avoided. In addition, a filter technique would be rapid, especially when multiple sampling is necessary, for it does not require continuous manipulation of many small test tubes through numerous centrifugation steps. MATERIALS Culture
AND
METHODS
of Cells
Chlamydomonas reinhardtii Dangeard 137 C+ was synchronously grown at 20°C on high salt minimal medium (12) at 400 fc on a 12 : 12 light: dark cycle. Cells were harvested during the logarithmic phase of growth at hour 4 in the light. Chlorella sorokiniana Shihira and Krauss was grown at 29°C on inorganic medium (13) at 450 fc under a continuous light regime. Cells were harvested during the logarithmic phase of growth. Ochromonas danica Pringsheim was grown at 29°C on complete medium (14) either in total darkness or at 450 fc on a constant light regime. Light-grown cells were harvested during the logarithmic phase of growth. Dark-grown cells were harvested during the linear phase of growth. Olisthodiscus luteus Carter was synchronously grown on an artificial seawater medium (15) at 400 fc on a 12 : 12 light : dark cycle. Cells were harvested during the logarithmic phase of growth. Unfertilized eggs of the sea urchin Strongylocentrotus purpuratus Stimps were kindly supplied by Dr. David Fromson of McGill University. Cell Counts Haemocytometer. trotus were counted
Chlamydomonas,
Chlorella,
using a Levy haemocytometer.
and StrongylocenFor the counts on
574
CATTOLICO
AND
GIBBS
the algal cultures, approximately 50 ~1 of 5.0% formalin were added per milliliter of culture. Coulter counter. Cell counts on the Ochromonas and Olisthodiscus cultures were made with a model ZB, Coulter counter equipped with a 100~pm aperture. Olisthodiscus cells were suspended and appropriately diluted in growth medium for count determinations, whereas Ochromonas cells were diluted in Isoton (Coulter Electronics, Mississauga, Ontario) prior to counting. Fluorometric
Analysis
of DNA
Materials and reagents. Metricel Alpha-6 cellulose filters, 25 mm in diameter and having a pore size of 0.45 pm, were purchased from Gelman Instrument Co., Ann Arbor, Michigan. Polyethylene BEEM capsules, size 00, with standard pyramidal tips, were purchased from Ladd Research Industries, Burlington, Vermont. Highly polymerized calf thymus DNA was purchased from Sigma Chemical Co., St. Louis, Missouri. A series of DNA stock solutions having a final concentration of 0, 0.125, 0.25, 0.5, and 0.75 pg/O.Ol ml were made using 1 M NH,OH. These solutions were stored at 5°C. Acetone, 100% ethanol, trichloroacetic acid, and HCl were of analytical grade. Norit neutral decolorizing charcoal was purchased from Fisher Scientific Co., Fairlawn, New Jersey. The fluorescent agent, 3,5-diaminobenzoic acid, lot lOlC-0330, was purchased from Sigma Chemical Co., St. Louis, Missouri. This material was purified immediately before use by adding 2.0 ml of 4 N HCl which was at 5°C to 0.6 g of the fluorescent agent. This mixture was agitated with a vortex mixer until all the 3,5-diaminobenzoic acid went into solution. The solution was then transferred to a 12-ml conical centrifuge tube containing approximately 20 mg of charcoal. The 3,5diaminobenzoic acid solution was carefully mixed with the charcoal by drawing it in and out of a Pasteur pipette. This careful mixing was done to avoid vigorous agitation which may cause (16) increased blank readings if applied after the initial thorough mixing of the DABA-HCI-charcoal solution. Following centrifugation at 2900 rpm for 5 min at 20°C in a clinical table-top centrifuge, the supernatant was removed with a Pasteur pipette and transferred to a second conical centrifuge tube containing 20 mg of charcoal. The DABA solution was again carefully mixed with the charcoal. The number of charcoal extractions needed for stable blank readings seems to vary from lot to lot of the fluorescent agent. For example, lot 10 1C-03 30 required seven extractions. The 3,5-diaminobenzoic acid solution should be clear to straw yellow by the end of the extraction sequence. Apparatus. Figure 1 presents a schematic diagram of the filter apparatus used in this assay. The unit consists of a standard l-liter filter flask
MICROFLUOROMETRIC
VACUUM
DNA
FILTER
ASSAY
575
t-.C
FIG. 1. Schematic drawing of the assembly of Millipore filter unit and adaptors for use in the microfluorometric assay of DNA. (A) Clamp top: (B) stainless steel disk; (C) Metricel Alpha-6 filters; (D) Teflon disk; (E) Millipore base: (F) clamp bottom which is attached to the clamp top by means of three r/4 in. bolts and secured with wing nuts: (C) 1 liter filter flask.
(G), followed by a ground glass Millipore base (E), and a disk-shaped Teflon adaptor (D) on which the Metricel filters (C) rest. A Plexiglas clamp (A and F) holds the adaptors in place. Details of the stainless steel adaptor are presented in Fig. 2A. This adaptor is 5.7 cm wide and contains four sample troughs located 2.0 cm from the outer edge of the adaptor. The sample troughs are 0.6 cm in diameter and 0.55 cm deep. Figure 2B presents a detailed view of the Teflon adaptor. This disk is 5.7 cm wide and contains an 0 ring seated approximately 0.1 cm from the outer edge of the adaptor. An 0 ring is present on both the upper and lower sides of the disk. The four filter areas on this Teflon disk are located 2.0 cm from the outer edge of the disk and are formed by making a number of 0.64 mm holes in an area covering 0.28 cm*. The spaces between the microbore holes act as a support for the small-sized filters used. The clamp used to hold the adaptors in place is pictured in Fig. 3. This unit is made of Plexiglas and is held together by three l/4 in. bolts with wing nuts. Details of size specifications for this clamp are presented in the figure. A model 110 Turner Fluorometer equipped with a blue lamp, lamp adaptor, and high sensitivity sample holder was used. The primary filter was a Turner type 405 (excitation maximum 405 nm) whereas the secondary filter was a combination of Turner type 8 and 65A (emission maximum 520 nm). Cell sampling. A sample of cells was axenically removed from the cul-
576
CATTOLICO
AND
GIBBS
a
FIG. 2. Schematic diagrams of filter adaptors used in the microfluorometric assay of DNA. (A) Stainless steel disk which is 5.7 cm in diameter and 0.55 cm deep. This adaptor contains four holes, 0.6 cm in diameter, which are located 2.0 cm from the outer disk rim. (B) Teflon disk which is 5.7 cm in diameter and 0.5 cm deep. This adaptor contains four areas, 0.6 cm in diameter, which are covered with microbore holes, 0.64 mm in size. The drilled areas of this disk are located 2.0 cm from the outer edge. An 0 ring has been placed on both sides of this disk approximately 0.1 cm from the outer edge of the disk.
FIG. 3. Schematic drawing of the clamp used in the microfluorometric assay of DNA. Size specifications are presented in the figure. The top and bottom of this clamp unit are attached by means of three % in. bolts secured with wing nuts.
MICROFLUOROMETRIC
DNA
FILTER
ASSAY
577
ture. The sample size was dependent upon the culture source and cell concentration. Volumes larger than 0.8 ml were placed in a 12-ml conical centrifuge tube. A small quantity of 5.0% formalin was added to kill the cells. Samples were then centrifuged at 2900 rpm in a clinical table-top centrifuge. Chlamydomonas, Chlorella, and Ochromonas were centrifuged for 15 min whereas Olisthodiscus and Strongylocentrotus were centrifuged for a 5-min period. All but 0.5 ml of supernatant was removed and the cells were transferred to 6 x 50 mm Siliclad-coated test tubes. The conical 12-ml centrifuge tube was rinsed with two 0.2-ml washes of appropriate growth medium and these washes were also transferred to the 6 X 50-mm test tube. Cell samples which were 0.8 ml or smaller were directly placed into Siliclad-coated 6 X 50-mm test tubes. Cells were then centrifuged at 7000 rpm at 5°C in a Sorvall RC2-B centrifuge using an HB-4 swinging bucket rotor. Chlamydomonas, Chlorella, and Ochromonas were spun for 15 min whereas Olisthodiscus and Strongylocentrotus were centrifuged for an 8-min period. All but 50 ~1 of the supernatant was aspirated using a drawn-out Pasteur pipet. Cells were resuspended using a vortex mixer. This step was found to be critical for the proper extraction of cells in the following steps. To the resuspended pellet was added approximately 0.5 ml of 80% acetone in H,O. The sample was mixed and centrifuged as described above until the precipitate was colorless. All cell samples were extracted with acetone except Chlorella and Strongylocentrotus where 100% ethanol was used. Samples have been kept at 5°C under acetone for as long as a month with no detrimental effects to the fluorometric assay. Wash procedure. Cells which had been suspended in 80% acetone were transferred with a Pasteur pipet onto the Metricel filters which were mounted in the filter apparatus as described in Fig. 1. The cells were then washed twice using approximately 0.15 ml of 0.6 N trichloroacetic acid at 5°C for each wash. It must be noted that one wash was allowed to drain completely through the filter before the next wash was begun. This double wash was repeated using two washes of 0.15 ml of 5°C ethanol: water (2: 1) followed by two washes with 0.15 ml of 60°C ethanol: water (2: 1). The rate of suction should be relatively slow to avoid the possibility of rupturing the filters and to allow maximum exposure of the cells to the wash materials. The filters were then removed from the filter apparatus, placed in BEEM polyethylene capsules, and allowed to dry at 2O”C, usually overnight. Filters used for the standard curve were subject to the wash sequence described above. Calf thymus DNA dissolved in 1 N NH,OH was added to these filters after they had been placed into the BEEM capsules. When the purified DNA standard was added to the filter prior to the wash sequence, quantitative recoveries were not observed.
578
CATTOLICO
AND
GIBBS
Fluorescent assay. To each filter contained in a BEEM capsule was added 50 ~1 of the extracted 4 N HCl-DABA solution. The capsules were immediately sealed after the addition of this fluorescent agent and incubated in a 60°C water bath for 30 min. The capsules were allowed to cool at 20°C for 5 min after which 0.5 ml of 1 N HCl was added. Each sample was transferred to a 6 X 50-mm microcuvette and read in a Turner fluorometer as soon as the machine registered maximum fluorescence. The coefficient of variation between duplicate samples was calculated (17) by the formula CV = (S x 100/Y), where S and Y are equal to the standard deviation and the sample mean, respectively. RESULTS
A representative standard curve for the microfluorometric determination of DNA on cellulose filters is presented in Fig. 4. This figure shows a linear fluorescent response to the amount of DNA present. The assay is sensitive to DNA concentrations as low as 0.125 pg and is applicable over a large range of DNA concentrations. High blank readings were not observed using this filter method. Replicate samples were taken for each point on a standard curve. A mean coefficient of variation, calculated for the replicate samples of nine standard curves, was 3.3% with a standard deviation of + 1.2%. The standard deviation was calculated using arcsine values. It was important to determine whether the DNA concentration per cell obtained by this filter assay method resulted in DNA values similar to those obtained by the application of more classical macromethods. For this reason, the amount of DNA present in a number of cell types was measured by the microfluorometric filter technique. As seen in
FIG. 4. Standard curve for the fluorometric determination of DNA on Metricel Alpha-6 filters. Assay was run as described in Materials and Methods. Each point is the mean of two samples.
DNA
MICROFLUOROMETRIC
Physical
Organism Chlorella sorokiniana Chlamydomonas
579
ASSAY
TABLE 1 FILTER ASSAY OF CELLS HAVING DIFFERENT CHARACTERISTICS AND DNA CONTENT
MICROFLUOROMETRIC PHYSIOLOGICAL
Size OLm)
FILTER
characteristics
Presence of cell wall
DNA per cell (g x 10-13)
Growth conditions
Fluorometric analysis (Mean + SEM)
Literature value
Reference
3x4
+
Light
1.06 + 0.06
0.82”
(9T
reinhardtii Ochromonas
6.5 x 10
f
Light : dark
1.90 ? 0.12
1.24-2.01
(18, 19)
danicn
8.0 x 12
-
Light Dark
2.23 k 0.12 1.61 -c 0.02
80
-
2.89 2.31
c7.0) (20)
Strongylocentrotus purpuratus
(eggs)
31.4
k 2.9
33.0
(7)
a This is the 1 N amount of DNA. Our cells were grown asychronously and would be expected to have a slightly greater amount of DNA. * The strain of Chlorelia employed by these authors, C. pyrenoidosa strain 7- 1l-05, has been named C. sorokiniana by Shihira and Krauss (21).
Table
1, the values
concentration per cell for and Strongylocentrotus agree well with the values reported for these cells by other investigators. The fact that only small quantities of cells are required for the fluorescent determination of DNA by the filter technique should allow multiple sampling of a single culture over an extended time period. A 500-ml culture of the wall-less alga Olisthodiscus luteus was sampled every 45 min during the synchronous growth cycle. As seen in Fig. 5A, the amount of DNA in the culture remained constant until 1.5 hr into the dark phase of growth after which DNA synthesis began. DNA synthesis terminated at hour 10 in the dark. Cell division in the same culture did not begin until hour 4 in the dark and was completed by hour 3 in the light (Fig. 5B). In this experiment the amount of DNA in the culture increased by 80% whereas cell number increased by 83%. The average amount of DNA found in an Olisthodiscus cell at hour 6 in the light is 3.05 X lo-12g. Chlorella,
obtained
Chlamydomonas,
for DNA
Ochromonas,
DISCUSSION
In this communication we report upon a modification of the Kissane and Robins microfluorometric technique for the measurement of DNA. This new method allows the entire sequence of sample collection, wash, and incubation procedures to take place on cellulose filters. The choice of the proper filter was critical to the success of this assay. For example, both Millipore and glass fiber filters are unsatisfactory for these filters either dissolve in acetone or disintegrate on heating at 60°C
580
CATTOLICO
AND
GIBBS
FIG. 5. Determination of DNA and cell number over a synchronous cell cycle of Olisthodiscus luteus using the microfluorometric filter assay. Sampling and assay conditions are described in Materials and Methods. White bars at the top of this figure indicate the light periods of the cycle: black bars indicate dark periods. (A) DNA per milliliter of culture. Each point represents a single sample. (B) Number of cells per milliliter.
in the presence of mineral acid. The filter chosen, Metricel Alpha-6, is able to withstand exposure to a wash sequence of acetone, trichloroacetic acid, and ethanol, followed by an incubation at 60°C in the presence of 4 N HCl. A variety of cells were assayed for DNA content. These cells were all highly pigmented and were therefore subject to a prewash in acetone or ethanol before application to the filter. It should be noted that unpigmented cells such as mouse fibroblasts or human leukocytes could be suspended in acetone or ethanol and applied directly to the filter without prewashing. The cells studied in this work ranged in size from 3 x 4 pm for Chlorella to 80 pm for Strongylocentrotus eggs. These sea urchin eggs represent an example of the extreme upper limit of the capacity of this filter method to handle cells which have a large biomass to DNA ratio. Cells which had been collected on the cellulose filters were washed with trichloroacetic acid and ethanol. This wash sequence has been shown (1) effective in removing extraneous cellular materials which may react with 3,5-diaminobenzoic acid and give a fluorescent product. These washed cells, still on the filter, were placed in polyethylene BEEM capsules and allowed to dry at 20°C. Higher temperatures (i.e., 50-60°C) often used (22,23) in the fluorometric literature for drying extracted cell pellets caused a large variability between replicate filtercollected samples, especially when the filters were held a week or more at an elevated drying temperature. It is possible that filter decomposition
MICROFLUOROMETRIC
DNA
FILTER
ASSAY
581
might be initiated by this extended high temperature treatment for a light pink color begins to appear along the filter edges. After drying at 20°C the filters were saturated with 4 N HCl which contains the fluorescent agent and were then incubated at 60°C. The polyethylene BEEM capsules in which the filters rest serve as an ideal reaction chamber for this assay step. These capsules are approximately 0.6 ml in volume and seal tightly. The possibility of filter desiccation due to evaporation and the problem of condensation on the walls of the holding vessel are prevented by the use of such a small closed reaction chamber. It has been observed (22) that appreciable drift may occur in fluorometric readings during the analysis of a single set of experimental samples. This becomes an important factor when dealing with a fluorometric method wherein 30-40 samples plus lo-12 standards may be analysed in one experiment. Hinegardner (22) has reported that the use of 1 N HCl rather than 0.6 N perchloric acid for dilution of the final fluorescent product would eliminate this unstable condition. Our data support this observation. No appreciable change in fluorescent values was observed during the reading of any large-scale experiment. However, it was noted that the fluorescence in a single sample will decay quite rapidly while being read. It is therefore suggested that all experimental samples be read as soon as they reach maximum fluorescent yield. Radioactive precursor incorporation, surface fluorometry using intercalating dyes, and phosphorescence analysis have provided alternate micromethods to the classical diphenylamine and indole reactions for the measurement of DNA. However, these methods require cells which are actively synthesizing DNA (24), highly purified DNA preparations (25), or the use of evenly dispersed cell homogenates which have been predigested with one or more enzyme types (26). The modified Kissane and Robins’ microfluorometric technique reported here allows the quantitative measurement of DNA per cell as a culture progresses through each phase of a normal cell cycle without the need of separating that DNA from RNA or protein cellular components. In addition to this high degree of specificity, this filter method has the advantage of extreme sensitivity. The fact that so few cells are required for a DNA determination allows an entire cycle sequence study to be made using a single culture as a sample source. The applicability of this assay to synchronous growth studies is well demonstrated in our cycle analysis of the wall-less alga Olisthodistus luteus (Figs. 5A and 5B). ACKNOWLEDGMENTS We wish to thank Mr. John Boothroyd study. We also which to thank Mr. Keith US.
and Mr. Morgan
Douglas Liot for their assistance in this for making the fluorometer available to
582
CATTOLICO
AND
GIBBS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22, 23. 24. 25. 26.
Kissane, J. M., and Robins, E. (1958)J. Biol. Chem. 233, 184-188. Munro, H. N., and Fleck, A. (1966) Methods Biochem. Anal. 14, 113-175. Hutchison, W. C., and Munro, H. N. (1961) Analysr 86, 768-813. Ogur, M., and Rosen, G. (1950) Arch. Biochem. 25, 262-276. Schmidt, G., and Thannhauser, J. J. (1945) .I. Biol. Chem. 161, 83-89. Schneider, W. C. (1945)5. Bioi. Chem. 161,293-303. Giudice, G. (1973) Developmental Biology of the Sea Urchin Embryo, p. 201, Academic Press, New York. Burton, K. (1968) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12B, pp. 163-169, Academic Press, New York. Hopkins, H. A., Flora, J. B., and Schmidt, R. R. (1972) Arch. Biochen. Biophys. 153, 845-849. Santoianni, P., and Ayala, M. (1965) J. Invest. Dermatol. 45, 99-103. Switzer, B. R., and Summer, G. K. (1971) C/in. Chim. Acta 32, 203-206. Sueoka, N., Chiang, K. S., and Kates, J. R. (1967) J. Mol. Biol. 25, 47-66. Marvin, J. W., and Karlander, E. P. (1974) J. Phycol. 10, 24-28. Aaronson, S., and Baker, H. (1959) J. Prorozool. 6, 282-284. Cattolico, R. A., Boothroyd. J. C., and Gibbs, S. P., Plant Physiol., in press. Robertson, F. W., and Tait, K. (1971) Anal. Biochem. 41,477-481. Sokal, R. R., and Rohlf, F. J. (1969) Biometry, p. 62, W. H. Freeman and Co., San Francisco. Sueoka, N., Chiang, K. S., and Kates, J. R. (1967) J. Mol. Bio/. 25, 47-66. Kates, J. R., Chiang, K. S., and Jones, R. F. (1968) Exp. Cell Res. 49, 121-13.5. Gibbs, S. P., Mak, R., Ng, R., and Slankis, T. (1974) J. Cell Sci. 16, 579-591. Shihira, I., and Krauss, R. W. (1965) Chlorella, p. 30, Port City Press, Baltimore. Hinegardner, R. T. (1971) Anal. Biochem. 39, 197-201. Holm-Hansen, O., Sutcliffe, W. H., and Sharp, J. (1968) Limnot. Oceanogr. 13, 507-5 14. Hayashi, F., Ishida, M. R., and Kikuchi, T. (1969) Annu. Rep. Res. Reactor Inst. Kyoto Univ. 2, 56-66. Sheridan, R. E., O’Donnell, C. M., and Pautler, E. L. (1973) Anal. Biochem. 52, 657-659. Karsten, U., and Wollenberger, A. (1972) Anal. Biochem. 46, 135-148.
BIOCHEMISTRY
Rapid
Filter
69, 572-582
Method Analysis
(1975)
for the Microfluorometric of DNA’
ROSE ANN CATTOLICO~ AND SARAH P. GIBBS Department Montreal,
of Biology, McGill University, Quebec, Canada H3C 3Gl
Received June 10, 1975; accepted July 22, 1975 A rapid filter method for the microfluorometric analysis of DNA is reported in this communication. Cells collected on cellulose filters are subject to an assay sequence developed from a fluorometric method initially described by J. M. Kissane and E. Robins ((1958) J. Biol. Chem. 233, 184-188) for DNA quantitation. The assay is one of high specificity requiring no separation of DNA from other major cellular components. Samples containing as little as 0.2-0.3 pg of DNA have been analyzed by this filter method with a coefficient of variation for replicate standard samples of 3.3%. The DNA content of a number of cell types having different physiological characteristics has been determined by the use of this filter technique. The data obtained for Chlorella, Chlamydomonas, Ochromonas, and Stronglyocentrotus eggs were well within the values reported for these cells by other investigators using classical macromethods. We have taken advantage of the high specificity, sensitivity, and accuracy of this filter assay to determine DNA content during the synchronous growth cycle of the wall-less alga Olisthodiscus luteus using a single small volume culture as a sample source.
In 1958 Kissane and Robins (1) described a microfluorometric assay for the measurement of DNA. This assay requires the collection of microquantities of cells, a wash sequence to remove extraneous fluorescent materials, and finally an incubation at 60°C with 3,5-diaminobenzoic acid (DABA) dissolved in 4 N HCl. During the incubation step, the deoxyribose sugars released by the hydrolysis of the purine deoxyribonucleotides react in a highly specific manner with DABA to give a fluorescent product. The quantitation of DNA by this fluorometric assay has a number of technical advantages. Difficulties of nucleic acid degradation or incomplete recovery often encountered (see 2 and 3 for review) by the application of more classical DNA extraction methods (4-6) can be entirely avoided. Moreover, due to the high specificity of the fluorometric response, the presence of polysaccharides, protein, RNA, arabinose, and other substances which have been shown (2,7-9) i Supported by the National Research Council of Canada (Grant A-2921) and the Quebec Department of Education. * Present address: Department of Botany, University of Washington, Seattle, Washington 98195. 572 Copyri,ght All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
DNA
MICROFLUOROMETRIC
FILTER
ASSAY
573
to seriously affect the calorimetric and/or optical density measurement of DNA no longer presents a problem. It is interesting to note that although this fluorometric method has many obvious advantages, it has been rarely chosen by investigators. The tedious nature of microsample collection and manipulation has no doubt restricted its use. In this communication we wish to report upon a rapid method whereby the entire fluorometric assay sequence described above takes place on cellulose filters. The use of filters in the fluorometric determination of DNA would eliminate the need for precision in the pipetting of microquantities of cells (10) and wash materials (1). Cell adhesion to test tube surfaces during initial sample collection or during sample resuspension (11) would be eliminated. The tedious removal of microquantities of wash materials ( 1,lO) and therefore the possibility of sample loss during this often repeated step would be entirely avoided. In addition, a filter technique would be rapid, especially when multiple sampling is necessary, for it does not require continuous manipulation of many small test tubes through numerous centrifugation steps. MATERIALS Culture
AND
METHODS
of Cells
Chlamydomonas reinhardtii Dangeard 137 C+ was synchronously grown at 20°C on high salt minimal medium (12) at 400 fc on a 12 : 12 light: dark cycle. Cells were harvested during the logarithmic phase of growth at hour 4 in the light. Chlorella sorokiniana Shihira and Krauss was grown at 29°C on inorganic medium (13) at 450 fc under a continuous light regime. Cells were harvested during the logarithmic phase of growth. Ochromonas danica Pringsheim was grown at 29°C on complete medium (14) either in total darkness or at 450 fc on a constant light regime. Light-grown cells were harvested during the logarithmic phase of growth. Dark-grown cells were harvested during the linear phase of growth. Olisthodiscus luteus Carter was synchronously grown on an artificial seawater medium (15) at 400 fc on a 12 : 12 light : dark cycle. Cells were harvested during the logarithmic phase of growth. Unfertilized eggs of the sea urchin Strongylocentrotus purpuratus Stimps were kindly supplied by Dr. David Fromson of McGill University. Cell Counts Haemocytometer. trotus were counted
Chlamydomonas,
Chlorella,
using a Levy haemocytometer.
and StrongylocenFor the counts on
574
CATTOLICO
AND
GIBBS
the algal cultures, approximately 50 ~1 of 5.0% formalin were added per milliliter of culture. Coulter counter. Cell counts on the Ochromonas and Olisthodiscus cultures were made with a model ZB, Coulter counter equipped with a 100~pm aperture. Olisthodiscus cells were suspended and appropriately diluted in growth medium for count determinations, whereas Ochromonas cells were diluted in Isoton (Coulter Electronics, Mississauga, Ontario) prior to counting. Fluorometric
Analysis
of DNA
Materials and reagents. Metricel Alpha-6 cellulose filters, 25 mm in diameter and having a pore size of 0.45 pm, were purchased from Gelman Instrument Co., Ann Arbor, Michigan. Polyethylene BEEM capsules, size 00, with standard pyramidal tips, were purchased from Ladd Research Industries, Burlington, Vermont. Highly polymerized calf thymus DNA was purchased from Sigma Chemical Co., St. Louis, Missouri. A series of DNA stock solutions having a final concentration of 0, 0.125, 0.25, 0.5, and 0.75 pg/O.Ol ml were made using 1 M NH,OH. These solutions were stored at 5°C. Acetone, 100% ethanol, trichloroacetic acid, and HCl were of analytical grade. Norit neutral decolorizing charcoal was purchased from Fisher Scientific Co., Fairlawn, New Jersey. The fluorescent agent, 3,5-diaminobenzoic acid, lot lOlC-0330, was purchased from Sigma Chemical Co., St. Louis, Missouri. This material was purified immediately before use by adding 2.0 ml of 4 N HCl which was at 5°C to 0.6 g of the fluorescent agent. This mixture was agitated with a vortex mixer until all the 3,5-diaminobenzoic acid went into solution. The solution was then transferred to a 12-ml conical centrifuge tube containing approximately 20 mg of charcoal. The 3,5diaminobenzoic acid solution was carefully mixed with the charcoal by drawing it in and out of a Pasteur pipette. This careful mixing was done to avoid vigorous agitation which may cause (16) increased blank readings if applied after the initial thorough mixing of the DABA-HCI-charcoal solution. Following centrifugation at 2900 rpm for 5 min at 20°C in a clinical table-top centrifuge, the supernatant was removed with a Pasteur pipette and transferred to a second conical centrifuge tube containing 20 mg of charcoal. The DABA solution was again carefully mixed with the charcoal. The number of charcoal extractions needed for stable blank readings seems to vary from lot to lot of the fluorescent agent. For example, lot 10 1C-03 30 required seven extractions. The 3,5-diaminobenzoic acid solution should be clear to straw yellow by the end of the extraction sequence. Apparatus. Figure 1 presents a schematic diagram of the filter apparatus used in this assay. The unit consists of a standard l-liter filter flask
MICROFLUOROMETRIC
VACUUM
DNA
FILTER
ASSAY
575
t-.C
FIG. 1. Schematic drawing of the assembly of Millipore filter unit and adaptors for use in the microfluorometric assay of DNA. (A) Clamp top: (B) stainless steel disk; (C) Metricel Alpha-6 filters; (D) Teflon disk; (E) Millipore base: (F) clamp bottom which is attached to the clamp top by means of three r/4 in. bolts and secured with wing nuts: (C) 1 liter filter flask.
(G), followed by a ground glass Millipore base (E), and a disk-shaped Teflon adaptor (D) on which the Metricel filters (C) rest. A Plexiglas clamp (A and F) holds the adaptors in place. Details of the stainless steel adaptor are presented in Fig. 2A. This adaptor is 5.7 cm wide and contains four sample troughs located 2.0 cm from the outer edge of the adaptor. The sample troughs are 0.6 cm in diameter and 0.55 cm deep. Figure 2B presents a detailed view of the Teflon adaptor. This disk is 5.7 cm wide and contains an 0 ring seated approximately 0.1 cm from the outer edge of the adaptor. An 0 ring is present on both the upper and lower sides of the disk. The four filter areas on this Teflon disk are located 2.0 cm from the outer edge of the disk and are formed by making a number of 0.64 mm holes in an area covering 0.28 cm*. The spaces between the microbore holes act as a support for the small-sized filters used. The clamp used to hold the adaptors in place is pictured in Fig. 3. This unit is made of Plexiglas and is held together by three l/4 in. bolts with wing nuts. Details of size specifications for this clamp are presented in the figure. A model 110 Turner Fluorometer equipped with a blue lamp, lamp adaptor, and high sensitivity sample holder was used. The primary filter was a Turner type 405 (excitation maximum 405 nm) whereas the secondary filter was a combination of Turner type 8 and 65A (emission maximum 520 nm). Cell sampling. A sample of cells was axenically removed from the cul-
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a
FIG. 2. Schematic diagrams of filter adaptors used in the microfluorometric assay of DNA. (A) Stainless steel disk which is 5.7 cm in diameter and 0.55 cm deep. This adaptor contains four holes, 0.6 cm in diameter, which are located 2.0 cm from the outer disk rim. (B) Teflon disk which is 5.7 cm in diameter and 0.5 cm deep. This adaptor contains four areas, 0.6 cm in diameter, which are covered with microbore holes, 0.64 mm in size. The drilled areas of this disk are located 2.0 cm from the outer edge. An 0 ring has been placed on both sides of this disk approximately 0.1 cm from the outer edge of the disk.
FIG. 3. Schematic drawing of the clamp used in the microfluorometric assay of DNA. Size specifications are presented in the figure. The top and bottom of this clamp unit are attached by means of three % in. bolts secured with wing nuts.
MICROFLUOROMETRIC
DNA
FILTER
ASSAY
577
ture. The sample size was dependent upon the culture source and cell concentration. Volumes larger than 0.8 ml were placed in a 12-ml conical centrifuge tube. A small quantity of 5.0% formalin was added to kill the cells. Samples were then centrifuged at 2900 rpm in a clinical table-top centrifuge. Chlamydomonas, Chlorella, and Ochromonas were centrifuged for 15 min whereas Olisthodiscus and Strongylocentrotus were centrifuged for a 5-min period. All but 0.5 ml of supernatant was removed and the cells were transferred to 6 x 50 mm Siliclad-coated test tubes. The conical 12-ml centrifuge tube was rinsed with two 0.2-ml washes of appropriate growth medium and these washes were also transferred to the 6 X 50-mm test tube. Cell samples which were 0.8 ml or smaller were directly placed into Siliclad-coated 6 X 50-mm test tubes. Cells were then centrifuged at 7000 rpm at 5°C in a Sorvall RC2-B centrifuge using an HB-4 swinging bucket rotor. Chlamydomonas, Chlorella, and Ochromonas were spun for 15 min whereas Olisthodiscus and Strongylocentrotus were centrifuged for an 8-min period. All but 50 ~1 of the supernatant was aspirated using a drawn-out Pasteur pipet. Cells were resuspended using a vortex mixer. This step was found to be critical for the proper extraction of cells in the following steps. To the resuspended pellet was added approximately 0.5 ml of 80% acetone in H,O. The sample was mixed and centrifuged as described above until the precipitate was colorless. All cell samples were extracted with acetone except Chlorella and Strongylocentrotus where 100% ethanol was used. Samples have been kept at 5°C under acetone for as long as a month with no detrimental effects to the fluorometric assay. Wash procedure. Cells which had been suspended in 80% acetone were transferred with a Pasteur pipet onto the Metricel filters which were mounted in the filter apparatus as described in Fig. 1. The cells were then washed twice using approximately 0.15 ml of 0.6 N trichloroacetic acid at 5°C for each wash. It must be noted that one wash was allowed to drain completely through the filter before the next wash was begun. This double wash was repeated using two washes of 0.15 ml of 5°C ethanol: water (2: 1) followed by two washes with 0.15 ml of 60°C ethanol: water (2: 1). The rate of suction should be relatively slow to avoid the possibility of rupturing the filters and to allow maximum exposure of the cells to the wash materials. The filters were then removed from the filter apparatus, placed in BEEM polyethylene capsules, and allowed to dry at 2O”C, usually overnight. Filters used for the standard curve were subject to the wash sequence described above. Calf thymus DNA dissolved in 1 N NH,OH was added to these filters after they had been placed into the BEEM capsules. When the purified DNA standard was added to the filter prior to the wash sequence, quantitative recoveries were not observed.
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Fluorescent assay. To each filter contained in a BEEM capsule was added 50 ~1 of the extracted 4 N HCl-DABA solution. The capsules were immediately sealed after the addition of this fluorescent agent and incubated in a 60°C water bath for 30 min. The capsules were allowed to cool at 20°C for 5 min after which 0.5 ml of 1 N HCl was added. Each sample was transferred to a 6 X 50-mm microcuvette and read in a Turner fluorometer as soon as the machine registered maximum fluorescence. The coefficient of variation between duplicate samples was calculated (17) by the formula CV = (S x 100/Y), where S and Y are equal to the standard deviation and the sample mean, respectively. RESULTS
A representative standard curve for the microfluorometric determination of DNA on cellulose filters is presented in Fig. 4. This figure shows a linear fluorescent response to the amount of DNA present. The assay is sensitive to DNA concentrations as low as 0.125 pg and is applicable over a large range of DNA concentrations. High blank readings were not observed using this filter method. Replicate samples were taken for each point on a standard curve. A mean coefficient of variation, calculated for the replicate samples of nine standard curves, was 3.3% with a standard deviation of + 1.2%. The standard deviation was calculated using arcsine values. It was important to determine whether the DNA concentration per cell obtained by this filter assay method resulted in DNA values similar to those obtained by the application of more classical macromethods. For this reason, the amount of DNA present in a number of cell types was measured by the microfluorometric filter technique. As seen in
FIG. 4. Standard curve for the fluorometric determination of DNA on Metricel Alpha-6 filters. Assay was run as described in Materials and Methods. Each point is the mean of two samples.
DNA
MICROFLUOROMETRIC
Physical
Organism Chlorella sorokiniana Chlamydomonas
579
ASSAY
TABLE 1 FILTER ASSAY OF CELLS HAVING DIFFERENT CHARACTERISTICS AND DNA CONTENT
MICROFLUOROMETRIC PHYSIOLOGICAL
Size OLm)
FILTER
characteristics
Presence of cell wall
DNA per cell (g x 10-13)
Growth conditions
Fluorometric analysis (Mean + SEM)
Literature value
Reference
3x4
+
Light
1.06 + 0.06
0.82”
(9T
reinhardtii Ochromonas
6.5 x 10
f
Light : dark
1.90 ? 0.12
1.24-2.01
(18, 19)
danicn
8.0 x 12
-
Light Dark
2.23 k 0.12 1.61 -c 0.02
80
-
2.89 2.31
c7.0) (20)
Strongylocentrotus purpuratus
(eggs)
31.4
k 2.9
33.0
(7)
a This is the 1 N amount of DNA. Our cells were grown asychronously and would be expected to have a slightly greater amount of DNA. * The strain of Chlorelia employed by these authors, C. pyrenoidosa strain 7- 1l-05, has been named C. sorokiniana by Shihira and Krauss (21).
Table
1, the values
concentration per cell for and Strongylocentrotus agree well with the values reported for these cells by other investigators. The fact that only small quantities of cells are required for the fluorescent determination of DNA by the filter technique should allow multiple sampling of a single culture over an extended time period. A 500-ml culture of the wall-less alga Olisthodiscus luteus was sampled every 45 min during the synchronous growth cycle. As seen in Fig. 5A, the amount of DNA in the culture remained constant until 1.5 hr into the dark phase of growth after which DNA synthesis began. DNA synthesis terminated at hour 10 in the dark. Cell division in the same culture did not begin until hour 4 in the dark and was completed by hour 3 in the light (Fig. 5B). In this experiment the amount of DNA in the culture increased by 80% whereas cell number increased by 83%. The average amount of DNA found in an Olisthodiscus cell at hour 6 in the light is 3.05 X lo-12g. Chlorella,
obtained
Chlamydomonas,
for DNA
Ochromonas,
DISCUSSION
In this communication we report upon a modification of the Kissane and Robins microfluorometric technique for the measurement of DNA. This new method allows the entire sequence of sample collection, wash, and incubation procedures to take place on cellulose filters. The choice of the proper filter was critical to the success of this assay. For example, both Millipore and glass fiber filters are unsatisfactory for these filters either dissolve in acetone or disintegrate on heating at 60°C
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FIG. 5. Determination of DNA and cell number over a synchronous cell cycle of Olisthodiscus luteus using the microfluorometric filter assay. Sampling and assay conditions are described in Materials and Methods. White bars at the top of this figure indicate the light periods of the cycle: black bars indicate dark periods. (A) DNA per milliliter of culture. Each point represents a single sample. (B) Number of cells per milliliter.
in the presence of mineral acid. The filter chosen, Metricel Alpha-6, is able to withstand exposure to a wash sequence of acetone, trichloroacetic acid, and ethanol, followed by an incubation at 60°C in the presence of 4 N HCl. A variety of cells were assayed for DNA content. These cells were all highly pigmented and were therefore subject to a prewash in acetone or ethanol before application to the filter. It should be noted that unpigmented cells such as mouse fibroblasts or human leukocytes could be suspended in acetone or ethanol and applied directly to the filter without prewashing. The cells studied in this work ranged in size from 3 x 4 pm for Chlorella to 80 pm for Strongylocentrotus eggs. These sea urchin eggs represent an example of the extreme upper limit of the capacity of this filter method to handle cells which have a large biomass to DNA ratio. Cells which had been collected on the cellulose filters were washed with trichloroacetic acid and ethanol. This wash sequence has been shown (1) effective in removing extraneous cellular materials which may react with 3,5-diaminobenzoic acid and give a fluorescent product. These washed cells, still on the filter, were placed in polyethylene BEEM capsules and allowed to dry at 20°C. Higher temperatures (i.e., 50-60°C) often used (22,23) in the fluorometric literature for drying extracted cell pellets caused a large variability between replicate filtercollected samples, especially when the filters were held a week or more at an elevated drying temperature. It is possible that filter decomposition
MICROFLUOROMETRIC
DNA
FILTER
ASSAY
581
might be initiated by this extended high temperature treatment for a light pink color begins to appear along the filter edges. After drying at 20°C the filters were saturated with 4 N HCl which contains the fluorescent agent and were then incubated at 60°C. The polyethylene BEEM capsules in which the filters rest serve as an ideal reaction chamber for this assay step. These capsules are approximately 0.6 ml in volume and seal tightly. The possibility of filter desiccation due to evaporation and the problem of condensation on the walls of the holding vessel are prevented by the use of such a small closed reaction chamber. It has been observed (22) that appreciable drift may occur in fluorometric readings during the analysis of a single set of experimental samples. This becomes an important factor when dealing with a fluorometric method wherein 30-40 samples plus lo-12 standards may be analysed in one experiment. Hinegardner (22) has reported that the use of 1 N HCl rather than 0.6 N perchloric acid for dilution of the final fluorescent product would eliminate this unstable condition. Our data support this observation. No appreciable change in fluorescent values was observed during the reading of any large-scale experiment. However, it was noted that the fluorescence in a single sample will decay quite rapidly while being read. It is therefore suggested that all experimental samples be read as soon as they reach maximum fluorescent yield. Radioactive precursor incorporation, surface fluorometry using intercalating dyes, and phosphorescence analysis have provided alternate micromethods to the classical diphenylamine and indole reactions for the measurement of DNA. However, these methods require cells which are actively synthesizing DNA (24), highly purified DNA preparations (25), or the use of evenly dispersed cell homogenates which have been predigested with one or more enzyme types (26). The modified Kissane and Robins’ microfluorometric technique reported here allows the quantitative measurement of DNA per cell as a culture progresses through each phase of a normal cell cycle without the need of separating that DNA from RNA or protein cellular components. In addition to this high degree of specificity, this filter method has the advantage of extreme sensitivity. The fact that so few cells are required for a DNA determination allows an entire cycle sequence study to be made using a single culture as a sample source. The applicability of this assay to synchronous growth studies is well demonstrated in our cycle analysis of the wall-less alga Olisthodistus luteus (Figs. 5A and 5B). ACKNOWLEDGMENTS We wish to thank Mr. John Boothroyd study. We also which to thank Mr. Keith US.
and Mr. Morgan
Douglas Liot for their assistance in this for making the fluorometer available to
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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22, 23. 24. 25. 26.
Kissane, J. M., and Robins, E. (1958)J. Biol. Chem. 233, 184-188. Munro, H. N., and Fleck, A. (1966) Methods Biochem. Anal. 14, 113-175. Hutchison, W. C., and Munro, H. N. (1961) Analysr 86, 768-813. Ogur, M., and Rosen, G. (1950) Arch. Biochem. 25, 262-276. Schmidt, G., and Thannhauser, J. J. (1945) .I. Biol. Chem. 161, 83-89. Schneider, W. C. (1945)5. Bioi. Chem. 161,293-303. Giudice, G. (1973) Developmental Biology of the Sea Urchin Embryo, p. 201, Academic Press, New York. Burton, K. (1968) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12B, pp. 163-169, Academic Press, New York. Hopkins, H. A., Flora, J. B., and Schmidt, R. R. (1972) Arch. Biochen. Biophys. 153, 845-849. Santoianni, P., and Ayala, M. (1965) J. Invest. Dermatol. 45, 99-103. Switzer, B. R., and Summer, G. K. (1971) C/in. Chim. Acta 32, 203-206. Sueoka, N., Chiang, K. S., and Kates, J. R. (1967) J. Mol. Biol. 25, 47-66. Marvin, J. W., and Karlander, E. P. (1974) J. Phycol. 10, 24-28. Aaronson, S., and Baker, H. (1959) J. Prorozool. 6, 282-284. Cattolico, R. A., Boothroyd. J. C., and Gibbs, S. P., Plant Physiol., in press. Robertson, F. W., and Tait, K. (1971) Anal. Biochem. 41,477-481. Sokal, R. R., and Rohlf, F. J. (1969) Biometry, p. 62, W. H. Freeman and Co., San Francisco. Sueoka, N., Chiang, K. S., and Kates, J. R. (1967) J. Mol. Bio/. 25, 47-66. Kates, J. R., Chiang, K. S., and Jones, R. F. (1968) Exp. Cell Res. 49, 121-13.5. Gibbs, S. P., Mak, R., Ng, R., and Slankis, T. (1974) J. Cell Sci. 16, 579-591. Shihira, I., and Krauss, R. W. (1965) Chlorella, p. 30, Port City Press, Baltimore. Hinegardner, R. T. (1971) Anal. Biochem. 39, 197-201. Holm-Hansen, O., Sutcliffe, W. H., and Sharp, J. (1968) Limnot. Oceanogr. 13, 507-5 14. Hayashi, F., Ishida, M. R., and Kikuchi, T. (1969) Annu. Rep. Res. Reactor Inst. Kyoto Univ. 2, 56-66. Sheridan, R. E., O’Donnell, C. M., and Pautler, E. L. (1973) Anal. Biochem. 52, 657-659. Karsten, U., and Wollenberger, A. (1972) Anal. Biochem. 46, 135-148.