- Email: [email protected]
An ultramicro technique for the determination of deoxypentose nucleic acid
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
63,
446-151
(1956)
An Ultramicro Technique for the Determination of Deoxypentose Nucleic Acid’ Konrad Keck From the Department of Zoology, University of Wisconsin, Madison, Wisconsin
Received February 20, 1966 INTRODUCTION
We know of several methods, based on the color reaction of the sugar moiety, for the quantitative estimation of deoxypentose nucleic acid (DNA). The methods of Dische (1) with diphenylamine and of Stumpf (2) with cysteine hydrochloride are well known. Both methods permit the quantitative measurement of DNA in amounts of 25 to 250 pg./ml. Webb and Levy (3) obtained about the same sensitivity using the color reaction of DNA with p-nitrophenylhydrazine. Ceriotti (4)) on the other hand, has described a method which is about ten times more sensitive than the above-mentioned methods. His method is based upon the discovery of Dische (5) that indole gives a color reaction with DNA. This reaction, however, is also given by a number of other sugars, aliphatic aldehydes, and protein hydrolyzates in varying intensity; but the color produced by these substances is completely removed by repeated extractions with chloroform, while the color produced by DNA remains unaffected by this treatment. Ceriotti (4) found that arabinose behaved differently from the rest of the sugars in that the color was only partially extracted by chloroform and therefore interfered with the determination of DNA. This method permits the determination of DNA in amounts from 2.5 to 15 ~g./ml., and 2 ml. of solution is used for the test. The method of Ceriotti was found to be specially suited for an adaptation to the microliter
scale because of the simplicity
of the procedure and
the sensitivity of the reaction. In the present paper a modification is described which allows the estimation of DNA in amounts of 0.1-l pg. present in 20 ~1.solution. It has been possible to eliminate the unspecific * This work has been carried out under a fellowship of the U. S. Foreign Operations Administration, Scientists Research Project, TA 01-101-3006. 446
DETERMINATION
OF DNA
447
reaction given by arabinose, so that the modified method can also be used successfully for plant material. EXPERIMENTAL
Several alterations of the original method were found necessary in order to adapt the procedure with reasonable accuracy and simplicity to the microliter scale. The procedure had to be carried out in small tubes with a maximal capacity of 100 ~1. One of the main difficulties to overcome was the necessity for repeated extractions with chloroform after hydrolysis of the reaction mixture as described by Ceriotti. The chloroform collects at the bottom of the container after extraction and is rather difficult to remove from these small tubes without appreciable loss of the extracted solution. It was further observed that chloroform, which had been purified as described by Ceriotti, occasionally caused a slight but measurable fading of the color on storage. When several organic solvents were tested for their ability to replace chloroform, amyl acetate was found to be most practical. Amy1 acetate is almost as active in extracting only the unspecific colors, needs no special purification before use, has a lower water solubility than chloroform, and, in addition, due to its lower density, it floats on top of the aqueous solution and can easily be removed with a small pipet. A thorough mixing of the aqueous and the organic phase in 100~~1. tubes was achieved by means of a thin glass rod the end of which had been melted to form a small ball which fitted tightly into the tubes. By moving this pestle up and down, a fairly good dispersion of the organic phase was obtained. The two phases separated readily again after some minutes so that centrifugation was found unnecessary. During preliminary experiments it was observed that by lowering the concentration of HCl, which was necessary for the color development in the indole reaction with DNA, the formation of unspecific colors was minimized to a much greater extent than the specific color. For a more detailed investigation of this effect the following method was applied: Several test tubes containing 2 ml. of test substance, 1 ml. of HCl of varying concentration (1 N, 2 N, 6 N, and approximately 12 N2) and 1 ml. of 0.0401,indole solution were prepared and placed in a boiling water bath for 10 min. The tubes were cooled and extracted twice with equal volumes of amyl acetate. The optical density of the extracted solutions was read in a Beckman DU spectrophotometer at 490 rnp against water using a lo-mm. cell. --~__-_-. _ .-_. -_-.- .-.- --. ~. z Concentrated HCl of density 1.19.
448
KONFZAD
KECK
Pentose nucleic acid, sucrose, arabinose, and casein hydrolyzate were tested in a concentrat,ion of 1000 pg./ml., and DNA in the concentration of 50 pg./ml.
The results of these experiments are given in Fig. 1. The influence of the HCl concentration upon the color development of some substances which are known to give a color reaction with the indole reagent is shown in comparison with the reaction of DNA under the same circumstances. The unspecific color reactions were suppressed sufficiently by using 2 or 3 N I!ICl, even though only two extractions with amyl acetate were made and the concentration of the interfering substances was 20 times higher than the concentration of DNA. It should be mentioned that the color given by arabinose, which is not extracted completely and interferes in the reaction of Ceriotti, is no longer present. The formation of a pink color is also strongly reduced when hydrolyzed samples containing lesser concentrations of HCl are kept at room temperature for some time. In 1.0 r
IZN
6N HCI CONCENTRATION
2N
1N
FIG. 1. The influence of the HCl concentration on the color development in the indole reaction. The optical density was read at 490 rnp using a lo-mm. cell. 1: DNA 50 rg./ml.; 2: sucrose 1 mg./ml.; 3: arahinose 1 mg./ml.; 4: ribonucleic acid 1 mg./mI.; 5: casein hydrolyzate 1 mg./ml.
DETERMISATION
OF
TABLE
449
DN4
I
The Influence of the Indole Concentration on the Color Development in the Indole Reaction with DNA (18 pg./ml.) and Sucrose (1000 pg./ml.)
Readings were mftde itt 490 rnp using & IO-mm. cell Indole concentration %
0.04 0.06 (0.08) saturrtted
Optical density SUCIOSe DNA
0.086 0.074 0.0.52
0.210 0.224 0.250
the finally adopted procedure a HCl concentration of 2.5 N was chosen. Another slight increase in specificity was obtained by raising the indole concentration. Solutions of 0.04 and 0.06 % and a saturated solution (in warm water) of about 0.08 % were tested. The results for DNA (18 pg./ml.) and sucrose (1000 pg./ml.) are given in Table I. Only one extraction with amyl acetate was performed here in order to demonstrate the reaction with sucrose. Since reproducibility at the highest indole concentration (saturated) was found to be unreliable, the next lower concentration (0.06 %) was chosen. In the microliter scale it is convenient to prepare a combined HClindole reagent by mixing 5 ml. 2.5 N HCl and 5 ml. 0.06 % indole solution. This reagent should be made up fresh every day and kept in a stoppered flask. No specially calibrated micropipet is necessary, since one volume of the test solution and an equal volume of the HCl-indole reagent are used for the reaction. In the present work pipets with a volume between 20 and 25 ~1. were used. An absorption cell for ultramicro analysis is described by Holter and Qvtrup (6). For the present work another type was developed (see Fig. 2) which permits serial measurements.
1 FIG.
2. cross section and vertical
projection
of
the
:rbsorption cell unit-.
450
KONRAD
GAMMA
KECK
DNA/20,UL
3. Graph indicating the relationship between concentration and optical density of the indole reaction with DNA.2.5 N HCl and 0.06% indole solution. The optical density is calculated for a lo-mm. cell; the concentration is given in Pg./20 4. FIQ.
Polymethyl methacrylate (Lucite) plates with a thickness of 5-3 mm. were cut in parts 25 X 75 mm. The exact thickness of each block was checked by measuring several regions with a micrometer. The block was discarded when the measurements differed more than 0.03 mm. (0.5%). Two rows of holes were drilled, each being 1.5 mm. in diameter. Square pieces of cover slips were cemented on one side of each hole forming the bottom of the absorption cell. Another piece was used to cover the filled cell. The extinction was read at 490 rnw. using an Aminco microphotometer and a Leiss monochromator. The optical density read at a given cell length is converted to a theoretical cell length of 10 mm.
To determine the relationship between the optical density of the color reaction and the concentration of DNA, aqueous solutions of both DNAS and hydrolyzed DNA* were measured according to the procedure described above. When solutions of hydrolyzed DNA in 5 % trichloroacetic acid (TCA) were used, no inhibition of the color reaction in the presence of TCA was noticed. However hydrolyzed DNA was found to give a linear relationship in a much wider concentration range than unhydrolyzed aqueous solutions. In the case of hydrolyzed DNA, Beer’s law is followed up to a concentration of 50 pg./ml. The method can thus be a Rat liver DNA. Hydrolyzed in 5% TCA for 20 min. at 90°C.
4
DETERMINATION
OF DNA
451
applied successfully for the quantitative determination of DNA in TCA extracts according to the method of Schneider (7). In Fig. 3 the optical density was plotted against the concentration of hydrolyzed DNA, as calculated for a lo-mm. cell. The concentration of DNA is given here in pg./20 ccl.,as used for the test. The above-described method was used in a study of DNA synthesis in haploid and diploid embryos of Rana pip&s, to be published elsewbere. The determination proved to be sensitive enough to allow measurements of DNA on single embryos of early stageswith a reproducibility of f3%. ACKNOWLEDGMENTS The author is very much indebted to Prof. H. Ris, Department of Zoology, University of Wisconsin, for his constant interest and advice in this work. Thanks are also expressed to Dr. Liselotte I. Hecht, McArdle Memorial Laboratory, for kindly supplying samples of DNA used in these studies. SUMMARY
A modification of Dische’s (5) reaction of DNA with indole is described, which permits the determination of 0.1-l pg. DNA in the ultramicro scale. The specificity of the original reaction could be increased so that the arabinose does not interfere with the reaction, REFERENCES 1. DISCHE, Z., Mikrochemie 8, 4 (1930). 2. STUMPF, P. K., J. Biol. Chem. 189, 367 (1947). 3. WEBB, J. M., AND LEVY, H. B., J. Biol. Chem. 213, 107 (1955). 4. CERIOTTI, G., J. Biol. Chem. 198, 297 (1952). 5. DISCHE, Z., Biochem. 2. 204, 431 (1929). 6. HOLTER, H., AND L$VTRUP, S., Compt. rend. trav. lab. Carlsberg, SBr. chim. 27, 27 (1949). 7. SCHNEIDER, W. C., J. Biol. Chem. 161, 293 (1949).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
63,
446-151
(1956)
An Ultramicro Technique for the Determination of Deoxypentose Nucleic Acid’ Konrad Keck From the Department of Zoology, University of Wisconsin, Madison, Wisconsin
Received February 20, 1966 INTRODUCTION
We know of several methods, based on the color reaction of the sugar moiety, for the quantitative estimation of deoxypentose nucleic acid (DNA). The methods of Dische (1) with diphenylamine and of Stumpf (2) with cysteine hydrochloride are well known. Both methods permit the quantitative measurement of DNA in amounts of 25 to 250 pg./ml. Webb and Levy (3) obtained about the same sensitivity using the color reaction of DNA with p-nitrophenylhydrazine. Ceriotti (4)) on the other hand, has described a method which is about ten times more sensitive than the above-mentioned methods. His method is based upon the discovery of Dische (5) that indole gives a color reaction with DNA. This reaction, however, is also given by a number of other sugars, aliphatic aldehydes, and protein hydrolyzates in varying intensity; but the color produced by these substances is completely removed by repeated extractions with chloroform, while the color produced by DNA remains unaffected by this treatment. Ceriotti (4) found that arabinose behaved differently from the rest of the sugars in that the color was only partially extracted by chloroform and therefore interfered with the determination of DNA. This method permits the determination of DNA in amounts from 2.5 to 15 ~g./ml., and 2 ml. of solution is used for the test. The method of Ceriotti was found to be specially suited for an adaptation to the microliter
scale because of the simplicity
of the procedure and
the sensitivity of the reaction. In the present paper a modification is described which allows the estimation of DNA in amounts of 0.1-l pg. present in 20 ~1.solution. It has been possible to eliminate the unspecific * This work has been carried out under a fellowship of the U. S. Foreign Operations Administration, Scientists Research Project, TA 01-101-3006. 446
DETERMINATION
OF DNA
447
reaction given by arabinose, so that the modified method can also be used successfully for plant material. EXPERIMENTAL
Several alterations of the original method were found necessary in order to adapt the procedure with reasonable accuracy and simplicity to the microliter scale. The procedure had to be carried out in small tubes with a maximal capacity of 100 ~1. One of the main difficulties to overcome was the necessity for repeated extractions with chloroform after hydrolysis of the reaction mixture as described by Ceriotti. The chloroform collects at the bottom of the container after extraction and is rather difficult to remove from these small tubes without appreciable loss of the extracted solution. It was further observed that chloroform, which had been purified as described by Ceriotti, occasionally caused a slight but measurable fading of the color on storage. When several organic solvents were tested for their ability to replace chloroform, amyl acetate was found to be most practical. Amy1 acetate is almost as active in extracting only the unspecific colors, needs no special purification before use, has a lower water solubility than chloroform, and, in addition, due to its lower density, it floats on top of the aqueous solution and can easily be removed with a small pipet. A thorough mixing of the aqueous and the organic phase in 100~~1. tubes was achieved by means of a thin glass rod the end of which had been melted to form a small ball which fitted tightly into the tubes. By moving this pestle up and down, a fairly good dispersion of the organic phase was obtained. The two phases separated readily again after some minutes so that centrifugation was found unnecessary. During preliminary experiments it was observed that by lowering the concentration of HCl, which was necessary for the color development in the indole reaction with DNA, the formation of unspecific colors was minimized to a much greater extent than the specific color. For a more detailed investigation of this effect the following method was applied: Several test tubes containing 2 ml. of test substance, 1 ml. of HCl of varying concentration (1 N, 2 N, 6 N, and approximately 12 N2) and 1 ml. of 0.0401,indole solution were prepared and placed in a boiling water bath for 10 min. The tubes were cooled and extracted twice with equal volumes of amyl acetate. The optical density of the extracted solutions was read in a Beckman DU spectrophotometer at 490 rnp against water using a lo-mm. cell. --~__-_-. _ .-_. -_-.- .-.- --. ~. z Concentrated HCl of density 1.19.
448
KONFZAD
KECK
Pentose nucleic acid, sucrose, arabinose, and casein hydrolyzate were tested in a concentrat,ion of 1000 pg./ml., and DNA in the concentration of 50 pg./ml.
The results of these experiments are given in Fig. 1. The influence of the HCl concentration upon the color development of some substances which are known to give a color reaction with the indole reagent is shown in comparison with the reaction of DNA under the same circumstances. The unspecific color reactions were suppressed sufficiently by using 2 or 3 N I!ICl, even though only two extractions with amyl acetate were made and the concentration of the interfering substances was 20 times higher than the concentration of DNA. It should be mentioned that the color given by arabinose, which is not extracted completely and interferes in the reaction of Ceriotti, is no longer present. The formation of a pink color is also strongly reduced when hydrolyzed samples containing lesser concentrations of HCl are kept at room temperature for some time. In 1.0 r
IZN
6N HCI CONCENTRATION
2N
1N
FIG. 1. The influence of the HCl concentration on the color development in the indole reaction. The optical density was read at 490 rnp using a lo-mm. cell. 1: DNA 50 rg./ml.; 2: sucrose 1 mg./ml.; 3: arahinose 1 mg./ml.; 4: ribonucleic acid 1 mg./mI.; 5: casein hydrolyzate 1 mg./ml.
DETERMISATION
OF
TABLE
449
DN4
I
The Influence of the Indole Concentration on the Color Development in the Indole Reaction with DNA (18 pg./ml.) and Sucrose (1000 pg./ml.)
Readings were mftde itt 490 rnp using & IO-mm. cell Indole concentration %
0.04 0.06 (0.08) saturrtted
Optical density SUCIOSe DNA
0.086 0.074 0.0.52
0.210 0.224 0.250
the finally adopted procedure a HCl concentration of 2.5 N was chosen. Another slight increase in specificity was obtained by raising the indole concentration. Solutions of 0.04 and 0.06 % and a saturated solution (in warm water) of about 0.08 % were tested. The results for DNA (18 pg./ml.) and sucrose (1000 pg./ml.) are given in Table I. Only one extraction with amyl acetate was performed here in order to demonstrate the reaction with sucrose. Since reproducibility at the highest indole concentration (saturated) was found to be unreliable, the next lower concentration (0.06 %) was chosen. In the microliter scale it is convenient to prepare a combined HClindole reagent by mixing 5 ml. 2.5 N HCl and 5 ml. 0.06 % indole solution. This reagent should be made up fresh every day and kept in a stoppered flask. No specially calibrated micropipet is necessary, since one volume of the test solution and an equal volume of the HCl-indole reagent are used for the reaction. In the present work pipets with a volume between 20 and 25 ~1. were used. An absorption cell for ultramicro analysis is described by Holter and Qvtrup (6). For the present work another type was developed (see Fig. 2) which permits serial measurements.
1 FIG.
2. cross section and vertical
projection
of
the
:rbsorption cell unit-.
450
KONRAD
GAMMA
KECK
DNA/20,UL
3. Graph indicating the relationship between concentration and optical density of the indole reaction with DNA.2.5 N HCl and 0.06% indole solution. The optical density is calculated for a lo-mm. cell; the concentration is given in Pg./20 4. FIQ.
Polymethyl methacrylate (Lucite) plates with a thickness of 5-3 mm. were cut in parts 25 X 75 mm. The exact thickness of each block was checked by measuring several regions with a micrometer. The block was discarded when the measurements differed more than 0.03 mm. (0.5%). Two rows of holes were drilled, each being 1.5 mm. in diameter. Square pieces of cover slips were cemented on one side of each hole forming the bottom of the absorption cell. Another piece was used to cover the filled cell. The extinction was read at 490 rnw. using an Aminco microphotometer and a Leiss monochromator. The optical density read at a given cell length is converted to a theoretical cell length of 10 mm.
To determine the relationship between the optical density of the color reaction and the concentration of DNA, aqueous solutions of both DNAS and hydrolyzed DNA* were measured according to the procedure described above. When solutions of hydrolyzed DNA in 5 % trichloroacetic acid (TCA) were used, no inhibition of the color reaction in the presence of TCA was noticed. However hydrolyzed DNA was found to give a linear relationship in a much wider concentration range than unhydrolyzed aqueous solutions. In the case of hydrolyzed DNA, Beer’s law is followed up to a concentration of 50 pg./ml. The method can thus be a Rat liver DNA. Hydrolyzed in 5% TCA for 20 min. at 90°C.
4
DETERMINATION
OF DNA
451
applied successfully for the quantitative determination of DNA in TCA extracts according to the method of Schneider (7). In Fig. 3 the optical density was plotted against the concentration of hydrolyzed DNA, as calculated for a lo-mm. cell. The concentration of DNA is given here in pg./20 ccl.,as used for the test. The above-described method was used in a study of DNA synthesis in haploid and diploid embryos of Rana pip&s, to be published elsewbere. The determination proved to be sensitive enough to allow measurements of DNA on single embryos of early stageswith a reproducibility of f3%. ACKNOWLEDGMENTS The author is very much indebted to Prof. H. Ris, Department of Zoology, University of Wisconsin, for his constant interest and advice in this work. Thanks are also expressed to Dr. Liselotte I. Hecht, McArdle Memorial Laboratory, for kindly supplying samples of DNA used in these studies. SUMMARY
A modification of Dische’s (5) reaction of DNA with indole is described, which permits the determination of 0.1-l pg. DNA in the ultramicro scale. The specificity of the original reaction could be increased so that the arabinose does not interfere with the reaction, REFERENCES 1. DISCHE, Z., Mikrochemie 8, 4 (1930). 2. STUMPF, P. K., J. Biol. Chem. 189, 367 (1947). 3. WEBB, J. M., AND LEVY, H. B., J. Biol. Chem. 213, 107 (1955). 4. CERIOTTI, G., J. Biol. Chem. 198, 297 (1952). 5. DISCHE, Z., Biochem. 2. 204, 431 (1929). 6. HOLTER, H., AND L$VTRUP, S., Compt. rend. trav. lab. Carlsberg, SBr. chim. 27, 27 (1949). 7. SCHNEIDER, W. C., J. Biol. Chem. 161, 293 (1949).