Plant Molecular Biology Reporter 16: 183–189, 1998.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
Protocols
A Method for Isolation of Chloroplast DNA and
Mitochondrial DNA from Sunflower
S.O. TRIBOUSH∗, N.G. DANILENKO and O.G. DAVYDENKO
Institute of Genetics & Cytology, Department of Extranuclear Heredity, National Academy of
Sciences, 27 Academicheskaja str., 220072, Minsk, Belarus
Abstract. We present a method for isolation of chloroplast and mitochondrial DNA from
sunflower seedlings. The protocol includes: organelle isolation, deoxyribonuclease treatment,
lysis, deproteinisation and a final DNA purification with sodium dodecyl sulphate and potassium acetate. The organelle DNA yield is 5–10 micrograms per gram of tissue and the DNA
is fully restrictable. The technique is inexpensive and appropriate for the isolation of multiple
samples of organelle DNA from a small amount of tissue.
Key words: chloroplast DNA, mitochondrial DNA, organelle isolation, sunflower
Introduction
As part of our research on sunflower chlorina mutants, we undertook efforts
to extract DNA from the chloroplasts and mitochondria of sunflower. We first
considered the available methods (cpDNA: Herrmann, 1982; Bookjans et al.,
1984; Palmer, 1986; Maliga et al., 1995) (mtDNA: Crouzillat et al., 1987;
Köhler et al., 1991; Mackenzie, 1994). However, these were either very expensive (e.g. long-term gradient centrifugation) or they were not effective
when applied to our conditions. Consequently, we devised a new method
by combining elements from the other procedures with new steps based on
biochemical principles of DNA extraction. The following protocol is inexpensive, reproducible and can be used for extraction of both chloroplast and
mitochondrial DNA from sunflower seedlings.
∗ Author for correspondence. e-mail: [email protected]; fax: (0172) 63-58-27; ph:
(0172) 63-23-97.
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Materials and Methods
Solutions
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STE buffer: 400 mM sucrose, 50 mM Tris pH 7.8, 20 mM EDTA-Na2,
0.2% bovine serum albumin, 0.2% β-mercaptoethanol.
ST buffer: 400 mM sucrose, 50 mM Tris pH 7.8, 0.1% bovine serum
albumin.
TEN buffer: 100 mM Tris pH 7.2, 50 mM EDTA, 100 mM NaCl and
0.2% β-mercaptoethanol.
NETF buffer: 1.25 M NaCl, 50 mM EDTA, 50 mM Tris pH 8.0, 50 mM
NaF.
1 M Mg-acetate
5 M NH4 -acetate
5 M K-acetate
20% SDS
1 M EDTA-Na2
phenol (saturated with Tris, pH 8.0)
phenol:chlorophorm (1:1)
chloroform:isoamyl alcohol (24:1)
96% and 70% ethanol
isopropanol
DNase-1 (EC 3.1.21.1, DN-25, Lot 105H9556, Sigma)
Equipment
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mortar and pestle
refrigerated centrifuges (Beckman GPR, Beckman G2-21)
microfuge 11 (Beckman)
centrifuge tubes 50 ml
microcentrifuge tubes 1.5 ml
thermostat 37 ◦ C
waterbath 60 ◦ C, 65 ◦ C
−20 ◦ C freezer
Procedure
Chloroplast isolation
Green seedlings, kept in the dark for 48 h, are harvested, thoroughly rinsed,
cooled to 0 ◦ C, and ground in a cold mortar with STE buffer. The last two
components of the buffer are added just before the start of the experiment. All
the subsequent operations are conducted at 0 ◦ C unless otherwise specified.
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The homogenate is filtered through dense nylon and centrifuged (200 g). The
supernatant is then re-centrifuged (3700 g). Finally, the chloroplast pellet is
washed in the same buffer and re-pelleted (3700 g).
Protocol
1. Prepare STE buffer. Add BSA and β-mercaptoethanol just before use.
2. Prior to extraction, keep the plants in the dark for 48 h to reduce the starch
level. Harvest 5 g of 7–10 day-old, green seedlings. Rinse, and cool them
to 0 ◦ C. Conduct all subsequent operations on ice.
3. Homogenize the seedlings in the mortar with 20 ml of STE buffer.
4. Filter the homogenate through 50 µm nylon mesh and centrifuge the extract (Beckman GPR) at 1000 rpm (200 g) for 20 min.
5. Discard the nuclei pellet, centrifuge the supernatant at 4000 rpm (3700 g),
for 20 min.
6. Re-suspend the pellet gently by using a soft paintbrush, centrifuge at 4000
rpm for 20 min.
Mitochondria isolation
Dark-grown, etiolated seedlings are harvested and ground with STE buffer
the same way as for the chloroplast isolation. The subsequent steps are the
same as for the chloroplasts, except for the centrifugation rates: 4000 rpm for
pelleting of cell debris, nuclei and chloroplasts and 12,000 rpm (18,000 g) for
mitochondria pellets.
Protocol
1. The same as for Step I (chloroplast isolation).
2. Harvest 5 g of 7–10 day-old dark-grown etiolated seedlings, rinse and cool
them to 0 ◦ C. Conduct the subsequent operations on ice.
3–6. Same as the chloroplasts, except for the centrifugation rates: 4000 rpm
(step 4) and 12,000 rpm (18,000 g) Beckman G2-21 (Steps 5,6).
DNase treatment
The chloroplast or mitochondria pellet is homogenized with a soft paintbrush
in a minimal volume (not more than 0.2 mL) of ST buffer. The DNase-1
is freshly-dissolved in ST buffer with 0.02 M Mg-acetate and added to the
plant material to make a final concentration 25 µg/mL DNase-1 per g of
material. Both the DNase and Mg/EDTA-Na 2 ratios are essential for complete
removal of the nuclear DNA. Mg-acetate can be replaced by Mg-chloride
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or Mg-sulphate (Herrmann, 1982). The DNase treatment is done at 37 ◦ C
for 20 minutes (Crouzillat et al., 1987). The reaction is stopped by adding
EDTA-Na2 to a final concentration of 0.2 M.
DNase wash-off is performed in a special NETF buffer (Loeb and Chouvean, 1969). Chloroplasts or mitochondria are pelleted at appropriate speeds
for 20 min and lysed at once or stored frozen at −20 ◦ C.
Protocol
1. Re-suspend the organelle pellet in a minimal volume (not more than
0.2 mL) of ST buffer.
2. Dissolve 125 µg DNase-1 in 0.2 ml of ST buffer with 0.02 M Mg-acetate
and add to the organelle suspension. Adjust the final volume to 0.5 mL.
The final DNase concentration is 250 µg/mL.
3. Incubate at 37 ◦ C for 20 min; mix periodically.
4. Stop the reaction by adding EDTA-Na2 to the final concentration 0.2 M.
5. Wash the organelles from the DNase in 25–50 ml of NETF buffer (the
more buffer, the better). Collect the organelle pellets by centrifugation.
6. Keep the pellet at −20 ◦ C or lyse it at once.
Lysis and Purification
The organelle pellets are each re-suspended in TEN-buffer and lysed in 1%
SDS. If the tissue coalesces, the tubes are warmed in a 60 ◦ C water bath until the organelles are fully lysed. Phenol/chloroform deproteinization is then
carried out. The DNA pellet is washed repeatedly with ethanol (70%, 96%),
air-dried, and re-dissolved in TE-buffer.
Potassium acetate-SDS precipitation (according to the modified method
of Wilson and Chourey, 1984) is used for additional DNA cleaning. After
complete dissolving, the DNA is heated briefly with TEN buffer and SDS;
then potassium acetate is added. The mixture is shaken for a few min (until
the precipitate is dissolved), then it is frozen for 30 min. After centrifugation,
the supernatant is collected carefully to avoid transfer of pellet particles. The
supernatant is poured into ammonium acetate and isopropanol, mixed and
incubated at −20 ◦ C for 30 min. The precipitated DNA is then pelleted for 10
min in a microfuge (12,000 rpm), washed with ethanol (70%, 96%), dried at
room temperature, and re-dissolved in TE-buffer.
Protocol
1. Resuspend the pellet in 600 µL of TEN-buffer at 0 ◦ C and add 30 µL of
20% SDS by drops, constantly shaking the mixture.
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2. If the tissue coalesces, warm the tubes in a 60 ◦ C water bath until the
organelles are fully lysed.
3. Remove the proteins. Shake the mixture intensively for 5 min with an
equal volume of equilibrated phenol. Centrifuge for 10 min at 12,000 rpm
(microfuge 11). Remove the upper aqueous phase. Add phenol:chloroform
1:1. Repeat the procedure. Add chloroform:isoamyl alcohol 24:1. Repeat
the procedure.
4. Precipitate the DNA with 1/10 vol of 5 M ammonium acetate and 1 vol
of isopropanol at −20 ◦ C for 2–3 h or overnight. Centrifuge for 3–5 min
at 12,000 rpm. Wash the DNA pellet with 70% ethanol, then with pure
ethanol to remove residual salt.
5. Dry at room temperature and re-dissolve in TE-buffer (not more than
20 µL).
6. After the DNA is completely dissolved, add 150 µL of TEN buffer.
7. Incubate the DNA solution for 15 min at 65 ◦ C with 10 µL of 20% SDS.
Add 50 µL of 5 M potassium acetate. Shake the mixture at 65 ◦ C until the
precipitate dissolves, just a few minutes.
8. Incubate the DNA tube at −20 ◦ C for 30 min. Centrifuge for 10 min at
12,000 rpm.
9. Collect the supernatant carefully to avoid transfer of pellet particles (centrifuge the supernatant again if some of the particles are also transferred).
10. Pour the supernatant into 10 µL of 5 M ammonium acetate and 100 µL
isopropanol, mix and incubate at −20 ◦ C for 30 min.
11. Pellet the precipitated DNA for 10 min in a microfuge (12,000 rpm),
wash with ethanol (70%, 96%), dry at room temperature and re-dissolve
in TE-buffer.
12. Perform restriction enzyme digests as described by the manufacturer of
the enzymes (Ferments MBI).
Results
The DNA preparations from sunflower chloroplasts and mitochondria were
fully restrictable with 12 enzymes tested (unpublished data). Here we present
the results of cpDNA and mtDNA patterns digested by EcoR I (Figure 1).
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Figure 1. Ethidium bromide stained agarose gel of electrophoretically-separated sunflower
chloroplast DNA and mitochondrial DNA restriction fragments. λ/Hind, λDNA digested
with Hind III; cpDNA, undigested chloroplast DNA; mtDNA, undigested mitochondrial DNA;
cpDNA/EcoR I, chloroplast DNA digested by EcoR I; mtDNA/EcoR I, mitochondrial DNA
digested by EcoR I. Samples were size fractionated on 1% agarose gels containing 0.1 M
Tris-acetate (pH 8.0) and 1 mM EDTA. Electrophoresis was for 16 hr at 1 volt/cm.
Summary
Advantages and features of the above method
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Identical buffers are used for mitochondrial and chloroplast DNA isolation.
Only a small amount of material is required for DNA isolation (5 g of
plant tissue were enough for 20 endonuclease digests).
The DNA yield from sunflower seedlings was 5–10 µg/g of tissue.
Original use of NaF (the chemical inhibitor of DNase) for plant DNA
isolation.
The procedure does not require gradient ultracentrifugation (CsCl, sucrose, or percol) and involves only three short-time, differential, lowspeed centrifugations before lysis.
Original use of potassium acetate-SDS precipitation for cpDNA isolation. This guarantees restrictable mtDNA free from cpDNA and vice
versa.
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The DNA is usually of sufficient purity for restriction cleavage and blotting techniques.
The DNA solutions stored at −20 ◦ C are stable for at least a year without
any changes in their properties.
Acknowledgements
We thank Prof. D. Lando for critical review of the manuscript and A. Shymkevitch and V. Tsyvoon for technical assistance.
References
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