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A rapid procedure for isolating mitochondrial DNA
102 1988, Gene Anal Techn 5:102-104
___i- j
A Rapid Procedure for Isolating Mitochondrial DNA E A R L G. Z I M M E R M A N ,
of mammalian tissues. The technique eliminates the need for an ultracentrifuge and the use of cesium chloride gradients. Also, the technique permits the isolation of mtDNA from several individuals in a single day, decreasing the time for analysis of larger sample sizes from populations.
D A R R I N R. A K I N S , J O H N V. P L A N Z , and M I C H A E L J. S C H U R R
A technique for the rapid isolation of mitochondrial DNA (mtDNA) from animal tissues is described that eliminates the time-consuming separation of nuclear and mtDNAs using cesium chloride gradient ultracentrifugation. The procedure utilizes digestion of the nuclear DNA with DNase, after which lysis of mitochondria and subsequent extraction o f proteins results in relatively pure mtDNA. Up to 5 Ixg of mtDNA per gram of liver tissue resulted, a suitable yield for five digests with restriction enzymes and staining with ethidium bromide.
Studies of the rate of change and high levels of variation in mitochondrial D N A (mtDNA) offer an approach to the determination of lineages of populations, and the mtDNA genotypes of individual organisms can provide a definitive picture of female ancestry. Additionally, the complex and ambiguous nature of the nuclear genome, which makes it difficult to study, is not contained in m t D N A sequences. Recent investigations of mtDNA variation in natural populations have verified its usefulness as an evolutionary tool [1-3]. Nevertheless, the standard procedures for isolations of mtDNA are time consuming, involving separation of nuclear and mtDNA fractions by ultracentrifugation with cesium chloride gradients for 24-36 hours. Thus, for population studies where large numbers of organisms may be required, the time and expense of current techniques can be prohibitive for many research laboratories. Recently, various modified techniques have been described in an attempt to eliminate the use of cesium chloride gradients [4, 5]. Herein, a method is described for the rapid isolation of mtDNA from relatively small quantities
From the Department of Biological Sciences, University of North Texas, Denton. Address reprint requests to: Dr. Earl G. Zimmerman, Department of Biological Sciences, University of North Texas, P.O: Box 5218, Denton, TX 76203-5218. Received April 15, 1988.
Materials and Methods Deoxyribonuclease I, ribonuclease A, proteinase K, and various buffer reagents were obtained from Sigma Chemical Co. Restriction enzymes were purchased from Bethesda Research Laboratories.
Isolation of Mitochondria Standard sucrose gradient methodology was used to isolate a relatively pure mitochondrial fraction. Tissues (usually 2 - 3 g of liver) were homogenized in 15 ml of 240 mm Sucrose-1 mm EDTA (pH 7.4). This homogenate was layered over 20 ml of 340 mm sucrose, 1 mm EDTA (pH 7.4) and centrifuged at 700g for 15 minutes to remove cellular debris. The supernatant was decanted into a new centrifuge tube and centrifuged at 5,000g for 15 minutes to pellet mitochondria. The pellet containing mitochondria was carefully resuspended in 2 ml of 240 mm sucrose, 1 mm EDTA (pH 7.4); 15 ml of 240 mm sucrose, 1 mm EDTA (pH 7.4) were added, and the suspension was centrifuged at 5,000g for 15 minutes.
Removal of Nuclear DNA and Extraneous Protein The mitochondrial pellet was dispersed in 2.0 ml of 300 mm sucrose, 5 mm MgClz, 0.15% bovine serum albumin, 20 mm Tris-HC1 (pH 7.5), and 10 ~1 DNase I (1 mg/ml in 150 mm NaCI, 50% glycerol, stored at - 20°C) were added. Then, 10 p~l RNase A were added (10 mg/ml of 10 mm TrisHCI, 15 mm NaC1, pH 7.5, was heated to 100°C for 15 minutes, allowed to cool slowly to room temperature, and stored at -20°C) [6]. This mixture was incubated at 37°C for 30 minutes. After the addition of 4.0 ml of 300 mm sucrose, 5 mm MgCI2, 0.15% bovine serum albumin 20 mm Tris-HCl (pH 7.5), the mixture was centrifuged at 5,000g for 15 minutes. The supernatant was discarded, and this step was repeated. The resulting pellet contained nuclear DNAfree mitochondria. The mitochondrial pellet was resuspended in 2.0
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017 0735-0651/88/$03.50
to3 1988, Gene Anal Techn 5:102-104
ml 100 mm EDTA, 200 mm NaCI, 100 mm Tris (pH 8.0), 60 Izl of proteinase K (20 mg/ml) were added, and the mixture was incubated 5 minutes at room temperature. The mitochondria were then lysed by adding 50 Ixl of 10% SDS and incubated for 20 minutes at 37°C. The lysate was then centrifuged at 8,000g for 15 minutes to remove mitochondrial membranes. The resultant supernatant contained a suspension of mtDNA and some protein. Protein was extracted with an equal volume of distilled, buffered phenol [7] and centrifuged for 2 minutes at 10,000 rpm. The supernatant was decanted, and additional proteins were extracted twice with an equal volume of phenol:chloroform: isoamyl alcohol (24:1 chloroform:isoamyl alcohol, pH 7.6), and centrifuged for 2 minutes at 10,000 rpm. This step was repeated again until no protein was seen at the interface between the aqueous layer and phenol. The supernatant was decanted, and additional proteins were extracted with an equal volume of chloroform:isoamyl alcohol (24: I chloroform:isoamyl alcohol, pH 7.6). The supernatant was decanted, and traces of phenol and other organic substances were extracted with an e q u a l v o l u m e o f h y d r a t e d e t h y l e t h e r (1:1 water:ether) and centrifuged for 2 minutes at 10,000 rpm. The ether layer (top layer) was discarded, and the bottom layer contained mtDNA. The m t D N A was p r e c i p i t a t e d for 15-30 minutes at -70°C in a half volume of 7.5 M ammonium acetate and three volumes of ethanol and centrifuged at 10,000 rpm for 15 minutes to pellet the mtDNA. The ethanol/ammonium acetate was decanted, and the pellet was washed with 1 ml 70% ethanol to remove salt. The 70% ethanol was decanted, and the pellet was dried using vacuum centrifugation.
-j
_
Z
6000
4000
2000 0
I 1,0
I 2.0
I 3.0
I 4,0
1 5.0
I 6,0
g TISSUE Figure 1. Yields of DNA per gram of liver tissue from rodents using rapid isolation procedure for mtDNA.
Figure 2. Agarose gel comparing H i n c l I digests of liver mtDNA from two species of rodents and k phage D N A cut with Hindlll as a molecular weight standard. Lanes 3 and 4 are mtDNA from two rodent species using the rapid isolation procedure, lanes 2 and 5 are the mtDNAs of the two species isolated by cesium chloride gradient centrifugation, and lanes 1 and 6 are h phage DNA. /
/
j"
Restriction Digests and Electrophoresis The pellet was resuspended in 50-100 ixl TE buffer, and the yield of DNA was obtained spectrophotometrically to compare tissue quantities with mtDNA yield. Digestion with HinclI was done according to recommended procedures, and standard electrophoresis of restriction digests was run in a 0.7% agarose gel, 1.0 × TBE (8.9 mm Tris, 8.9 mm boric acid, 2 mm EDTA) electrode buffer, pH 8.0, at 35 V for 12 hours.
-----E_
1
2
3
4
5
6
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
f
J
104 1988, Gene Anal Techn 5:102-104
f-
Results and Discussion Isolated mtDNA restriction digests from this procedure appear free from any nuclear DNA contamination. In Figure 1, comparative results of H i n c l I digests of two species of rodent liver mtDNAs isolated using this technique (lanes 3 and 4) and those isolated in cesium chloride gradients (lanes 2 and 5) provided the same resulting patterns. Additionally, the yields of mtDNA from this technique are equal to or greater than those from the cesium chloride gradient technique. This is visible in the alternate restriction digests depicted in Figure 1. Yields of mtDNA plotted against weights of eight different tissue samples are shown in Figure 2. Yields ranged from 850-5,010 ~g mtDNA/ml from tissues weighing 0.65-6.0 g, respectively. The amount of mtDNA found in liver cells is a reflection of the physiologic condition of the animal. Stressed (unfed) animals produce more m t D N A than those receiving food ad libitum. Average yields of 5 ~g mtDNA per gram of liver were typical and are more than double those reported for small rodent livers using cesium chloride gradient [8]. Furthermore, yields are sufficient for as many as five digests from a single gram of tissue. Additionally, fragments can be detected by ethidium bromide staining, and it is not necessary to use radioactive labeling. The most important feature of the technique is the time saved by eliminating the use of ultracentrifugation. Previously, the mtDNA from only six animals could be isolated during an approximated 40-hour period, the limitation being the number of
spaces in the ultracentrifuge rotor. With this technique, the high-speed centrifuge rotor, with eight to 12 spaces depending on the model, is the limitation. However the limitation is not severe, and in a 6-hour period one person can process 16 animals up to the point of enzyme digestion. Additionally, the technique works well with small amounts of tissue, necessitated by many studies of small organisms for which pooling samples has been the only alternative.
This work was supported by grant 2 S07 RR07195 from the National Institutes of Health Biomedical Research Support Program and a Faculty Research Grant from the University of North Texas.
References 1. Lansman, R. A., Shade, R. O., Shapira, J. E, and Avise, J. C. (1981) J. Mol. Evol. 17,214-226. 2. Avise, J. C., and Lansman, R. A. (1983) in Evolution of Genes and Proteins (Nei, M., and Koehn, R. K.. eds.), pp. 147-164, Sinauer Assoc., Inc., Sunderland, MA. 3. Moritz, C., Dowling, T. E., and Brown, W. M. (1987) Ann. Rev. Ecol. Syst. 18,269-292. 4. Powell, J. R., and Zuniga, M. C. (1983) Biochem. Genet. 21, 1051-1055. 5. Chapman, R. W., and Powers, D. A. (1984) Tech. Rpt. UM-SG-TS-84-05 Maryland Sea Grant Prog., 1-11. 6. Maniatis, T., Fritsch, E. E, and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 7. Boehringer Mannheim (1987) Biochemica 4, 10. 8. Avise, J. C., Lansman, R. A., and Shade, R. O. (1979) Genetics 92, 279-295.
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
___i- j
A Rapid Procedure for Isolating Mitochondrial DNA E A R L G. Z I M M E R M A N ,
of mammalian tissues. The technique eliminates the need for an ultracentrifuge and the use of cesium chloride gradients. Also, the technique permits the isolation of mtDNA from several individuals in a single day, decreasing the time for analysis of larger sample sizes from populations.
D A R R I N R. A K I N S , J O H N V. P L A N Z , and M I C H A E L J. S C H U R R
A technique for the rapid isolation of mitochondrial DNA (mtDNA) from animal tissues is described that eliminates the time-consuming separation of nuclear and mtDNAs using cesium chloride gradient ultracentrifugation. The procedure utilizes digestion of the nuclear DNA with DNase, after which lysis of mitochondria and subsequent extraction o f proteins results in relatively pure mtDNA. Up to 5 Ixg of mtDNA per gram of liver tissue resulted, a suitable yield for five digests with restriction enzymes and staining with ethidium bromide.
Studies of the rate of change and high levels of variation in mitochondrial D N A (mtDNA) offer an approach to the determination of lineages of populations, and the mtDNA genotypes of individual organisms can provide a definitive picture of female ancestry. Additionally, the complex and ambiguous nature of the nuclear genome, which makes it difficult to study, is not contained in m t D N A sequences. Recent investigations of mtDNA variation in natural populations have verified its usefulness as an evolutionary tool [1-3]. Nevertheless, the standard procedures for isolations of mtDNA are time consuming, involving separation of nuclear and mtDNA fractions by ultracentrifugation with cesium chloride gradients for 24-36 hours. Thus, for population studies where large numbers of organisms may be required, the time and expense of current techniques can be prohibitive for many research laboratories. Recently, various modified techniques have been described in an attempt to eliminate the use of cesium chloride gradients [4, 5]. Herein, a method is described for the rapid isolation of mtDNA from relatively small quantities
From the Department of Biological Sciences, University of North Texas, Denton. Address reprint requests to: Dr. Earl G. Zimmerman, Department of Biological Sciences, University of North Texas, P.O: Box 5218, Denton, TX 76203-5218. Received April 15, 1988.
Materials and Methods Deoxyribonuclease I, ribonuclease A, proteinase K, and various buffer reagents were obtained from Sigma Chemical Co. Restriction enzymes were purchased from Bethesda Research Laboratories.
Isolation of Mitochondria Standard sucrose gradient methodology was used to isolate a relatively pure mitochondrial fraction. Tissues (usually 2 - 3 g of liver) were homogenized in 15 ml of 240 mm Sucrose-1 mm EDTA (pH 7.4). This homogenate was layered over 20 ml of 340 mm sucrose, 1 mm EDTA (pH 7.4) and centrifuged at 700g for 15 minutes to remove cellular debris. The supernatant was decanted into a new centrifuge tube and centrifuged at 5,000g for 15 minutes to pellet mitochondria. The pellet containing mitochondria was carefully resuspended in 2 ml of 240 mm sucrose, 1 mm EDTA (pH 7.4); 15 ml of 240 mm sucrose, 1 mm EDTA (pH 7.4) were added, and the suspension was centrifuged at 5,000g for 15 minutes.
Removal of Nuclear DNA and Extraneous Protein The mitochondrial pellet was dispersed in 2.0 ml of 300 mm sucrose, 5 mm MgClz, 0.15% bovine serum albumin, 20 mm Tris-HC1 (pH 7.5), and 10 ~1 DNase I (1 mg/ml in 150 mm NaCI, 50% glycerol, stored at - 20°C) were added. Then, 10 p~l RNase A were added (10 mg/ml of 10 mm TrisHCI, 15 mm NaC1, pH 7.5, was heated to 100°C for 15 minutes, allowed to cool slowly to room temperature, and stored at -20°C) [6]. This mixture was incubated at 37°C for 30 minutes. After the addition of 4.0 ml of 300 mm sucrose, 5 mm MgCI2, 0.15% bovine serum albumin 20 mm Tris-HCl (pH 7.5), the mixture was centrifuged at 5,000g for 15 minutes. The supernatant was discarded, and this step was repeated. The resulting pellet contained nuclear DNAfree mitochondria. The mitochondrial pellet was resuspended in 2.0
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017 0735-0651/88/$03.50
to3 1988, Gene Anal Techn 5:102-104
ml 100 mm EDTA, 200 mm NaCI, 100 mm Tris (pH 8.0), 60 Izl of proteinase K (20 mg/ml) were added, and the mixture was incubated 5 minutes at room temperature. The mitochondria were then lysed by adding 50 Ixl of 10% SDS and incubated for 20 minutes at 37°C. The lysate was then centrifuged at 8,000g for 15 minutes to remove mitochondrial membranes. The resultant supernatant contained a suspension of mtDNA and some protein. Protein was extracted with an equal volume of distilled, buffered phenol [7] and centrifuged for 2 minutes at 10,000 rpm. The supernatant was decanted, and additional proteins were extracted twice with an equal volume of phenol:chloroform: isoamyl alcohol (24:1 chloroform:isoamyl alcohol, pH 7.6), and centrifuged for 2 minutes at 10,000 rpm. This step was repeated again until no protein was seen at the interface between the aqueous layer and phenol. The supernatant was decanted, and additional proteins were extracted with an equal volume of chloroform:isoamyl alcohol (24: I chloroform:isoamyl alcohol, pH 7.6). The supernatant was decanted, and traces of phenol and other organic substances were extracted with an e q u a l v o l u m e o f h y d r a t e d e t h y l e t h e r (1:1 water:ether) and centrifuged for 2 minutes at 10,000 rpm. The ether layer (top layer) was discarded, and the bottom layer contained mtDNA. The m t D N A was p r e c i p i t a t e d for 15-30 minutes at -70°C in a half volume of 7.5 M ammonium acetate and three volumes of ethanol and centrifuged at 10,000 rpm for 15 minutes to pellet the mtDNA. The ethanol/ammonium acetate was decanted, and the pellet was washed with 1 ml 70% ethanol to remove salt. The 70% ethanol was decanted, and the pellet was dried using vacuum centrifugation.
-j
_
Z
6000
4000
2000 0
I 1,0
I 2.0
I 3.0
I 4,0
1 5.0
I 6,0
g TISSUE Figure 1. Yields of DNA per gram of liver tissue from rodents using rapid isolation procedure for mtDNA.
Figure 2. Agarose gel comparing H i n c l I digests of liver mtDNA from two species of rodents and k phage D N A cut with Hindlll as a molecular weight standard. Lanes 3 and 4 are mtDNA from two rodent species using the rapid isolation procedure, lanes 2 and 5 are the mtDNAs of the two species isolated by cesium chloride gradient centrifugation, and lanes 1 and 6 are h phage DNA. /
/
j"
Restriction Digests and Electrophoresis The pellet was resuspended in 50-100 ixl TE buffer, and the yield of DNA was obtained spectrophotometrically to compare tissue quantities with mtDNA yield. Digestion with HinclI was done according to recommended procedures, and standard electrophoresis of restriction digests was run in a 0.7% agarose gel, 1.0 × TBE (8.9 mm Tris, 8.9 mm boric acid, 2 mm EDTA) electrode buffer, pH 8.0, at 35 V for 12 hours.
-----E_
1
2
3
4
5
6
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
f
J
104 1988, Gene Anal Techn 5:102-104
f-
Results and Discussion Isolated mtDNA restriction digests from this procedure appear free from any nuclear DNA contamination. In Figure 1, comparative results of H i n c l I digests of two species of rodent liver mtDNAs isolated using this technique (lanes 3 and 4) and those isolated in cesium chloride gradients (lanes 2 and 5) provided the same resulting patterns. Additionally, the yields of mtDNA from this technique are equal to or greater than those from the cesium chloride gradient technique. This is visible in the alternate restriction digests depicted in Figure 1. Yields of mtDNA plotted against weights of eight different tissue samples are shown in Figure 2. Yields ranged from 850-5,010 ~g mtDNA/ml from tissues weighing 0.65-6.0 g, respectively. The amount of mtDNA found in liver cells is a reflection of the physiologic condition of the animal. Stressed (unfed) animals produce more m t D N A than those receiving food ad libitum. Average yields of 5 ~g mtDNA per gram of liver were typical and are more than double those reported for small rodent livers using cesium chloride gradient [8]. Furthermore, yields are sufficient for as many as five digests from a single gram of tissue. Additionally, fragments can be detected by ethidium bromide staining, and it is not necessary to use radioactive labeling. The most important feature of the technique is the time saved by eliminating the use of ultracentrifugation. Previously, the mtDNA from only six animals could be isolated during an approximated 40-hour period, the limitation being the number of
spaces in the ultracentrifuge rotor. With this technique, the high-speed centrifuge rotor, with eight to 12 spaces depending on the model, is the limitation. However the limitation is not severe, and in a 6-hour period one person can process 16 animals up to the point of enzyme digestion. Additionally, the technique works well with small amounts of tissue, necessitated by many studies of small organisms for which pooling samples has been the only alternative.
This work was supported by grant 2 S07 RR07195 from the National Institutes of Health Biomedical Research Support Program and a Faculty Research Grant from the University of North Texas.
References 1. Lansman, R. A., Shade, R. O., Shapira, J. E, and Avise, J. C. (1981) J. Mol. Evol. 17,214-226. 2. Avise, J. C., and Lansman, R. A. (1983) in Evolution of Genes and Proteins (Nei, M., and Koehn, R. K.. eds.), pp. 147-164, Sinauer Assoc., Inc., Sunderland, MA. 3. Moritz, C., Dowling, T. E., and Brown, W. M. (1987) Ann. Rev. Ecol. Syst. 18,269-292. 4. Powell, J. R., and Zuniga, M. C. (1983) Biochem. Genet. 21, 1051-1055. 5. Chapman, R. W., and Powers, D. A. (1984) Tech. Rpt. UM-SG-TS-84-05 Maryland Sea Grant Prog., 1-11. 6. Maniatis, T., Fritsch, E. E, and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 7. Boehringer Mannheim (1987) Biochemica 4, 10. 8. Avise, J. C., Lansman, R. A., and Shade, R. O. (1979) Genetics 92, 279-295.
© 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017