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An improved method for the analysis of the RNA-directed DNA polymerase reaction by polyacrylamide gels
ANALYTICAL
BIOCHEMISTRY
An Improved DNA
Method
Polymerase J. JUSTIN
Michigan
54,
Cancer
54-57 (1973)
for the Analysis Reaction
MCCORMICK Foundation,
Received September
of the RNA-Directed
by Polyacrylamide AND
42711 John
LENORA R. Street,
Gels
CALHOUN
Detroit,
Michigan
1, 1972; accepted January
16, 1973
@201
Analysis of the products of the RNA-directed DNA polymerase reaction using glycerol-substituted polyacrylamide gels has proven superior to the glycerol gradient method for quantitative assay of a series of nucleic acid samples. To overcome the phenomenon of a smearing of background radioactive counts in the gels caused by unincorporated ‘H-thymidine triphosphate, an automatic washing procedure making use of activated charcoal has been devised for the gels. This washing virtually eliminates all background while leaving the “H-DNA/35S RNA hybrid as well as the “HDNA/“H-DNA molecules fixed in situ on the gels.
Recently a system has been described for the detection of oncogenic viruses by the identification of a 70 S RNA hybridized to a newly synthesized radioactive DNA indicating the presence of an RNA-directed DNA polymerase (1). In an effort to simplify this technique for extension to the screening of large numbers of samples, we sought to analyze the hybrid product formed by such a reaction by electrophoresis on glycerolsubstituted polyacrylamide gels inst.ead of glycerol gradients. We found, however, that even after two alcohol precipitations of the nucleic acids, sufficient SH-thymidine triphosphate remains to run as a smear on glycerol-substituted gels obscuring the “H-DNA/RNA peak. To obviate this difficulty we have devised a washing procedure in which unincorporated 3H-thymidine triphosphate is removed from the gel while the 3H-DNA/RNA hybrid remains fixed in situ. An alternate approach to this problem was taken by Bishop et ,al. (2) who used Sephadex column chromatography to remove unincorporated nucleotides. We have found that the washing method described here is simpler for multiple samples of small volume. METHODS
The preparation of virus and the DNA polymerase reaction were carried out as described by Schlom and Spiegelman (1). After extraction of t,he reaction mixture with phenol-cresol, 50 pg of high molecular weight 54 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
ACRYLAMIDE-GEL
ANALYSIS
OF
3H-DNA/RNA
55
yeast RNA was added as carrier and the solution brought to 0.4 M with LiCI,. Two and a half volumes of ethanol were added and the nucleic acids precipitated overnight at -20°C. It was then centrifuged at 16,300g in the HB-4 head of a Sorvall centrifuge at 4°C for 30 min, the supernatant discarded, and the pellet re-dissolved in 50 ~1 of one half strength electrophoresis buffer containing bromphenol blue and 30% glycerol. This was applied to 1.8% polyacrylamide gels containing 0.5% agarose prepared according to the method of Peacock and Dingman (3) but with the addition of 10% reagent-grade glycerol. Electrophoresis was carried out in Tris-borate-EDTA buffer, pH 8.3, (3) at 2 mA/gel for 120 min at 4°C using a Hoefer electrophoresis unit. Reagents for gel electrophoresis were purchased from BioRad Laboratories, Richmond, Calif. At the end of the run, gels were removed from their tubes, transferred to 15 X 120-mm plastic test tubes perforated with small holes, and suspended for washing in a beaker containing 4 liters of 5 % acetic acid and 10% glycerol. The liquid was circulated through a 3-in. bed of charcoal pieces (approximately 1 cm” each) by means of a small aquarium pump (Dynaflow Motor Filter #425, Metaframe Corp., Maywood, N. J.) in a manner similar to the use of charcoal as a destaining aid with gels (4). The washed-gels were then removed, frozen at -20°C and cut in l-mm slices with a Mickle gel cutter. The gel slices were transferred to scintillation vials, digested with 0.3 ml of 30% H,O, for 1 hr at 60°C and counted on a Packard Liquid Scintillation Spectrometer after the addition of 5 ml of Aquasol. Counting efficiency was 16%. Decolorizing charcoal was prepared by washing 2 hr in 4 N KOH, 2 hr in 4 N HCl, and then overnight in running water. After it had been used for washing more than 200 gels, the charcoal was regenerated in the tank by washing with 7.4 N NH,OH for 7 hr and then washing overnight in running water to remove the nucleotides. RESULTS
AND
DISCUSSION
Figure 1A shows the profile of a gel containing 3H-DNA/35S RNA hybrid and the tritiated DNA/DNA product prepared from Rauscher leukemia virus and washed as described above. Figure 1B shows the profile of an identical preparation except that the gel was not washed. To determine whether this technique was as sensitive as the glycerol gradient technique, the reverse transcriptase reaction was carried out with Rauscher leukemia virus and the product split into two equal parts. Half was analyzed by the gel technique using the charcoal step and half analyzed by 10 to 30% glycerol gradients as described (1). Equivalent amounts of 3H-DNA/RNA hybrid and tritiated DNA/DNA product were found by each method. Also gel electrophoresis of tritiated RNA
56
MCCORMICK
AND
CALHOUN
B
4
3
300
"0
"0 200
l2
x
I
2
E
:: I
100
TOP
IO
20
30
40
SAMPLE
50
TOP
NUMBER
IO
20
30
40
50
f Imm.Slicec~
FIG. 1. Shows the radioactivity profiles of the products of the reverse transcriptase reaction carried out by the method of Schlom and Spicgelman (1) and analyzed by gel electrophoresis, A shows the profile of a gel washed according to the charcoal method described in the text. B shows the profile of a gel which was not washed. Mouse rRNA was analyzed on a separate gel to determine the S values. The peak in A at slice number 33 is the ‘H-DNA/35S RNA hybrid molecule formed in the reaction. The peak in A at slice number 46 is the tritiated DNA/DNA product of the reaction and has been characterized by its sensitivity to DNAase and by its buoyant density in Cs2S04 gradients.
and DNA prepared from mouse cells in culture gave equivalent results with or without the charcoal washing. This demonstrates that this method for analysis of products of the reverse transcriptsse react.ion is as sensitive as the glycerol gradient method for t,he analysis of products of the reverse transcriptase reaction and that no selective loss or degradation of product occurs with this method. Another mouse leukemia virus (5) and virions from human milk (6) gave results similar to those shown in Fig. IA. In preparations from each of these viruses, one finds 70s and/ or 35 S RNA indicating that, some preparations are more stable than others. This fact has also been noted by other workers (6). Noted added: We now find that electrophoresis at 2 mA per gel for 1 hr and sectioning of gels at 2 mm gives good resolution of 35/70 S RNA and the DNA/DNA product and saves a good deal of time. Ribosomal RNA marker must still be sectioned at 1 mm in order to see the separate 28 and 18 S peaks.
ACRYLAMIDE-GEL
ANALYSIS
OF
3H-DNA/RNA
ACKNOWLEDGMENTS We wish to express our appreciation to Dr. Arnold Dion of the Institute for Medical Research of Camdrn, N. J. who shared with us his experience with gel electrophorcsis of such DNA/RNA hyhrids and Dr. J. Schlom of the Institute of Cancer Research, Columbia Univrrsity, for his interest and advice in the use of the “H-DNA/RNA hybrid detection system for oncogenic viruses. We also thank Dr. James Arnold of this institute for providing lsbcled mouse cells and mouse leukemia virus for these esperimonts. The work was supported by Grant Special Virus Cancer Program Contract 712421 from the National Institutes of Health, USPH, and an Institutional Grant to the Michigan Cancer Foundation from the United Foundation of Greater Detroit. REFERENCES 1. SCHLOM, J. AND SPIECEL~MAN, S. (1971) Science 174, 840. 2. BISHOP, D. H. L., RWRECHT, R., SIMPSON, R. W. AND SPEIGELZVIMAN, S. (1971) J. Viral. 8, 730. 3. PEACOCK, A. C. AND DINGMMAN. C. W. (1968) Biochem. 7, 668. 4. GATHERCIHE, I,. J. AND KLEIN, L. (1971) Anal. Biochem. 44, 232. 5. SOULE, H. D. AND ARNOLD, W. J. (1970) J. Nat. Cancer Inst. 45, 253. 6. SCHLOM, J., SPIEGELMAN, S. AND MOORE, D. H. (1972) Science 175, 542.
BIOCHEMISTRY
An Improved DNA
Method
Polymerase J. JUSTIN
Michigan
54,
Cancer
54-57 (1973)
for the Analysis Reaction
MCCORMICK Foundation,
Received September
of the RNA-Directed
by Polyacrylamide AND
42711 John
LENORA R. Street,
Gels
CALHOUN
Detroit,
Michigan
1, 1972; accepted January
16, 1973
@201
Analysis of the products of the RNA-directed DNA polymerase reaction using glycerol-substituted polyacrylamide gels has proven superior to the glycerol gradient method for quantitative assay of a series of nucleic acid samples. To overcome the phenomenon of a smearing of background radioactive counts in the gels caused by unincorporated ‘H-thymidine triphosphate, an automatic washing procedure making use of activated charcoal has been devised for the gels. This washing virtually eliminates all background while leaving the “H-DNA/35S RNA hybrid as well as the “HDNA/“H-DNA molecules fixed in situ on the gels.
Recently a system has been described for the detection of oncogenic viruses by the identification of a 70 S RNA hybridized to a newly synthesized radioactive DNA indicating the presence of an RNA-directed DNA polymerase (1). In an effort to simplify this technique for extension to the screening of large numbers of samples, we sought to analyze the hybrid product formed by such a reaction by electrophoresis on glycerolsubstituted polyacrylamide gels inst.ead of glycerol gradients. We found, however, that even after two alcohol precipitations of the nucleic acids, sufficient SH-thymidine triphosphate remains to run as a smear on glycerol-substituted gels obscuring the “H-DNA/RNA peak. To obviate this difficulty we have devised a washing procedure in which unincorporated 3H-thymidine triphosphate is removed from the gel while the 3H-DNA/RNA hybrid remains fixed in situ. An alternate approach to this problem was taken by Bishop et ,al. (2) who used Sephadex column chromatography to remove unincorporated nucleotides. We have found that the washing method described here is simpler for multiple samples of small volume. METHODS
The preparation of virus and the DNA polymerase reaction were carried out as described by Schlom and Spiegelman (1). After extraction of t,he reaction mixture with phenol-cresol, 50 pg of high molecular weight 54 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
ACRYLAMIDE-GEL
ANALYSIS
OF
3H-DNA/RNA
55
yeast RNA was added as carrier and the solution brought to 0.4 M with LiCI,. Two and a half volumes of ethanol were added and the nucleic acids precipitated overnight at -20°C. It was then centrifuged at 16,300g in the HB-4 head of a Sorvall centrifuge at 4°C for 30 min, the supernatant discarded, and the pellet re-dissolved in 50 ~1 of one half strength electrophoresis buffer containing bromphenol blue and 30% glycerol. This was applied to 1.8% polyacrylamide gels containing 0.5% agarose prepared according to the method of Peacock and Dingman (3) but with the addition of 10% reagent-grade glycerol. Electrophoresis was carried out in Tris-borate-EDTA buffer, pH 8.3, (3) at 2 mA/gel for 120 min at 4°C using a Hoefer electrophoresis unit. Reagents for gel electrophoresis were purchased from BioRad Laboratories, Richmond, Calif. At the end of the run, gels were removed from their tubes, transferred to 15 X 120-mm plastic test tubes perforated with small holes, and suspended for washing in a beaker containing 4 liters of 5 % acetic acid and 10% glycerol. The liquid was circulated through a 3-in. bed of charcoal pieces (approximately 1 cm” each) by means of a small aquarium pump (Dynaflow Motor Filter #425, Metaframe Corp., Maywood, N. J.) in a manner similar to the use of charcoal as a destaining aid with gels (4). The washed-gels were then removed, frozen at -20°C and cut in l-mm slices with a Mickle gel cutter. The gel slices were transferred to scintillation vials, digested with 0.3 ml of 30% H,O, for 1 hr at 60°C and counted on a Packard Liquid Scintillation Spectrometer after the addition of 5 ml of Aquasol. Counting efficiency was 16%. Decolorizing charcoal was prepared by washing 2 hr in 4 N KOH, 2 hr in 4 N HCl, and then overnight in running water. After it had been used for washing more than 200 gels, the charcoal was regenerated in the tank by washing with 7.4 N NH,OH for 7 hr and then washing overnight in running water to remove the nucleotides. RESULTS
AND
DISCUSSION
Figure 1A shows the profile of a gel containing 3H-DNA/35S RNA hybrid and the tritiated DNA/DNA product prepared from Rauscher leukemia virus and washed as described above. Figure 1B shows the profile of an identical preparation except that the gel was not washed. To determine whether this technique was as sensitive as the glycerol gradient technique, the reverse transcriptase reaction was carried out with Rauscher leukemia virus and the product split into two equal parts. Half was analyzed by the gel technique using the charcoal step and half analyzed by 10 to 30% glycerol gradients as described (1). Equivalent amounts of 3H-DNA/RNA hybrid and tritiated DNA/DNA product were found by each method. Also gel electrophoresis of tritiated RNA
56
MCCORMICK
AND
CALHOUN
B
4
3
300
"0
"0 200
l2
x
I
2
E
:: I
100
TOP
IO
20
30
40
SAMPLE
50
TOP
NUMBER
IO
20
30
40
50
f Imm.Slicec~
FIG. 1. Shows the radioactivity profiles of the products of the reverse transcriptase reaction carried out by the method of Schlom and Spicgelman (1) and analyzed by gel electrophoresis, A shows the profile of a gel washed according to the charcoal method described in the text. B shows the profile of a gel which was not washed. Mouse rRNA was analyzed on a separate gel to determine the S values. The peak in A at slice number 33 is the ‘H-DNA/35S RNA hybrid molecule formed in the reaction. The peak in A at slice number 46 is the tritiated DNA/DNA product of the reaction and has been characterized by its sensitivity to DNAase and by its buoyant density in Cs2S04 gradients.
and DNA prepared from mouse cells in culture gave equivalent results with or without the charcoal washing. This demonstrates that this method for analysis of products of the reverse transcriptsse react.ion is as sensitive as the glycerol gradient method for t,he analysis of products of the reverse transcriptase reaction and that no selective loss or degradation of product occurs with this method. Another mouse leukemia virus (5) and virions from human milk (6) gave results similar to those shown in Fig. IA. In preparations from each of these viruses, one finds 70s and/ or 35 S RNA indicating that, some preparations are more stable than others. This fact has also been noted by other workers (6). Noted added: We now find that electrophoresis at 2 mA per gel for 1 hr and sectioning of gels at 2 mm gives good resolution of 35/70 S RNA and the DNA/DNA product and saves a good deal of time. Ribosomal RNA marker must still be sectioned at 1 mm in order to see the separate 28 and 18 S peaks.
ACRYLAMIDE-GEL
ANALYSIS
OF
3H-DNA/RNA
ACKNOWLEDGMENTS We wish to express our appreciation to Dr. Arnold Dion of the Institute for Medical Research of Camdrn, N. J. who shared with us his experience with gel electrophorcsis of such DNA/RNA hyhrids and Dr. J. Schlom of the Institute of Cancer Research, Columbia Univrrsity, for his interest and advice in the use of the “H-DNA/RNA hybrid detection system for oncogenic viruses. We also thank Dr. James Arnold of this institute for providing lsbcled mouse cells and mouse leukemia virus for these esperimonts. The work was supported by Grant Special Virus Cancer Program Contract 712421 from the National Institutes of Health, USPH, and an Institutional Grant to the Michigan Cancer Foundation from the United Foundation of Greater Detroit. REFERENCES 1. SCHLOM, J. AND SPIECEL~MAN, S. (1971) Science 174, 840. 2. BISHOP, D. H. L., RWRECHT, R., SIMPSON, R. W. AND SPEIGELZVIMAN, S. (1971) J. Viral. 8, 730. 3. PEACOCK, A. C. AND DINGMMAN. C. W. (1968) Biochem. 7, 668. 4. GATHERCIHE, I,. J. AND KLEIN, L. (1971) Anal. Biochem. 44, 232. 5. SOULE, H. D. AND ARNOLD, W. J. (1970) J. Nat. Cancer Inst. 45, 253. 6. SCHLOM, J., SPIEGELMAN, S. AND MOORE, D. H. (1972) Science 175, 542.