Molecular Techniques and Methods

Isolation of Mitochondrial DNA
from Cell Suspension Cultures

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Mitochondria is separated by differential centrifugation from the bulk of nuclei, plastids, and cellular debris which differ in particle size or density. Mitochondria are then further separated from the remaining plastids and nuclear debris by gradient centrifugation. Homogenization media for mitochondrial isolation contain an osmoticum, EDTA, bovine serum albumin (BSA), 2-mercaptoethanol, and polyvinylpyrrolidone (PVP) which binds phenolics.

Suspension culture is a useful source of nongreen tissue in small-seeded species lacking fleshy fruit, large storage organs, or convenient nongreen floral tissue. This strategy should be considered for species in which axenic vegetative tissue is not easily obtained, or if green tissues do not yield mtDNA.

  • Yield of mtDNA; 50-100 ug/ 100 g of packed suspension culture cells.





  • MATERIALS AND SOLUTIONS

    Bead-Beater
    This device breaks cells by agitating them with glass beads in a small plastic vessel, and is simpler and less expensive than a French press.


    Suspension Culture Grinding Buffer (1 liter)
    0.3 M Mannitol ----------------------------------- 54.66 g
    50 mM Tris-HCl (pH 8.0) ------------------------ 50 ml of 1 M Tris-HCl
    3 mM EDTA ------------------------------------- 6 ml of 0.5 M EDTA
    0.1% BSA --------------------------------------- 1 g
    20 mM 2-mercaptoethanol ----------------------- 1.4 ml of 14.4 M 2-mercaptoethanol
    Deionized H2O to make a final volume of -------- 1 liter


    DNase I (10 mg/ml)


    Gradient Buffer (1 liter)
    0.3 M Sucrose ------------------------------------ 102.7 g
    50 mM Tris-HCl (pH 8.0) ------------------------ 50 ml of 1 M Tris-HCl
    20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
    0.1% BSA ---------------------------------------- 1 g
    Deionized H2O to make a final volume of -------- 1 liter


    1.6 M Sucrose Step Gradient Buffer (100 ml)
    1.6 M Sucrose ------------------------------------ 54.8 g
    50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
    20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
    0.1% BSA ---------------------------------------- 0.1 g
    Deionized H2O to make a final volume of -------- 100 ml


    1.2 M Sucrose Step Gradient Buffer (100 ml)
    1.2 M Sucrose ------------------------------------ 41.1 g
    50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
    20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
    0.1% BSA ---------------------------------------- 0.1 g
    Deionized H2O to make a final volume of -------- 100 ml


    0.6 M Sucrose Step Gradient Buffer (100 ml)
    0.6 M Sucrose ------------------------------------ 20.5 g
    50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
    20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
    0.1% BSA ---------------------------------------- 0.1 g
    Deionized H2O to make a final volume of -------- 100 ml


    ET buffer (100 ml)
    20 mM EDTA ------------------------------------- 4 ml of 0.5 M EDTA
    50 mM Tris-HCl (pH 8.0) ------------------------- 5 ml of 1 M Tris-HCl
    Deionized H2O ------------------------------------ 91 ml




    PROCEDURES

    Isolation of Mitochondria

  • All steps should be performed in ice!

    1. Grow suspension cultures in UMIA medium in dim light until cultures appear white or light yellow and creamy. Such cultures usually have lower amounts of phenolic compounds so that 2-mercaptoethanol is sufficient and PVP can be omitted from the Suspension Culture Grinding Buffer. If phenolics are present in damaging quantities, 1.0% PVP can be included during homogenization.

    2. Break suspension cultures in a Bead-Beater with ice in outer jacket. Three times for 10 second-pulse at high speed.
  • If using a French Press, break suspension cultures at 3,000 psi.

    3. Collect 400-500g cells by centrifugation at 1,500g for 10 minutes at 4oC.

    4. Resuspend cells 1:1 (v/v) in ice-cold Suspension Culture Grinding Buffer.

    5. Pour the suspension into a Bead-Beater vessel containing one-half volume of 500-um glass beads.

    6. Break suspension cultures in a Bead-Beater with ice in outer jacket. Three times for 10 second-pulse at high speed.

    7. Decant the disrupted cells from the glass beads into a Miracloth-lined funnel.

    8. Centrifuge the filtered preparation for 10 minutes at 1,500g to remove cell debris, plastids, and nuclei.

    9. Transfer the supernatant to a new centrifuge tube. Centrifuge at 15,000g for 15 minutes at 4oC.

    10. Resuspend the brownish mitochondrial pellet in about 25 ml of Suspension Culture Grinding Buffer.

    11. Add 300 ul of 1 M MgCl2 and 250 ul of DNase I (10 mg/ml) to the crude mitochondrial suspension.

    12. Incubate for 30 minutes on ice.

    13. Add 1 ml 0.5 M EDTA to the incubation medium to stop the reaction.

    14. Wash the mitochondria by diluting to about 400 ml with Gradient Buffer and centrifuging at 15,000 g for 15 minutes.

    15. Resuspend the pellet in 10 ml of Gradient Buffer.

    16. Layer 3-4 ml of mitochondrial suspension per sucrose step gradient consisting of 10 ml 1.6 M Sucrose Step Gradient Buffer, 10 ml 1.2 M Sucrose Step Gradient Buffer, and 10 ml 0.6 M Sucrose Step Gradient Buffer.

    17. Centrifuge for 1 hour at 25,000 rpm for purification from contaminating subcellular structures and residual DNase.

    18. A cream colored band will appear at both interfaces. Collect only the lower band, which contains most of the mtDNA, with a syringe.

    19. Dilute the mitochondrial fraction with 3 volumes of Gradient Buffer slowly over a 15-minute period to minimize disruption by osmotic shock.

    20. Harvest the mitochondria by centrifugation at 15,000g for 10 minutes at 4oC.

    21. Resuspend the mitochondrial pellet in 7 ml of ET buffer.

    22. Add 350 ul of 10% Sarkosyl to lyse the mitochondria.
  • Optional; add 1 ul of Proteinase K (20 mg/ml) and incubate for 15 minutes on ice to reduce clumps of debris.


    CsCl Gradients for mtDNA Purification

    23. To 7 ml of lysed mitochondria, add 8.05 g CsCl and 220 ul of ethidium bromide (10 mg/ml).

    24. Add ET buffer to make the total weight to 8.32 g. Dissolve the CsCl.

    25. Transfer the liquid to a Beckman Quick-Seal tube and heat-seal the tube.

    26. Centrifuge at 65,000 rpm for 8-10 hours at 20oC.

    27. Collect DNA band with a syringe.
  • In some preparations, particularly with certain genotypes, a lower band of supercoiled mtDNA is also present. Collect supercoiled mtDNA, also.

    28. Extract the ethidium bromide three times with isopropanol equilibrated with CsCl-saturated TE buffer.

    29. Dialyze mtDNA against 4 liters of STE buffer for 1-2 hour.

    30. Add 1/10th volume of 3 M Sodium acetate (pH 7.0) and 2.5 volume of ethanol.

    31. Precipitate DNA at -20oC overnight.

    32. Collect DNA by centrifugation at 15,000 rpm at 4oC for 20 minutes.

    33. Resuspend mtDNA in 100-500 ul TE and store at -20oC or 4oC.




    NOTES

  • Deoxyribonuclease (DNase) is used to remove remaining DNA external to the mitochondria. The effectiveness of this DNase step requires penetration of the DNase into nonintact contaminating plastids and nuclear debris and sufficient mitochondrial integrity to prevent the enzyme from entering the organelles. Integrity of mitochondria is critical when utilizing a DNase step during purification. This treatment should be performed early in the isolation procedure while the highest proportion of mitochondria are intact.


  • In some mtDNA preparations, faint bands which comigrate with purified plastid DNA restriction fragments can be seen on ethidium bromide-stained gels of restriction enzyme-digested mtDNA preparations. Whether these represent plastid DNA contamination can be checked by hybridizing total plastid DNA (nick-translated) to a Southern blot.


  • If DNase treatment of sucrose gradient-purified fractions does not give adequate purification from plastid DNA, dyes which enhance separation of organelle DNAs can be incorporated into the CsCl gradients. Plastid DNA could be quantitatively separated from mtDNA on CsCl gradients containing diamidinophenylindol or bisbenzimide.





  • KIT INFORMATION




    REFERENCES

  • Boeshore, ML, Lifshitz, I, Hanson, MR, Izhar, S (1983) Mol. Gen. Genet. 190: 459.


  • Clark, EM, Hanson, MR (1983) Plant Mol. Biol. Rep. 1: 77.


  • DeBonte, LR, Matthews, BF (1984) Plant Mol. Biol. Rep. 2: 32.


  • Dixon, LK, Leaver, CJ (1982) Plant Mol. Biol. 1: 89.


  • Galun, E, Arzee-Gonen, P, Fluhr, R, Edelman, M, Aviv, D (1982) Mol. Gen. Genet. 176: 50.


  • Hack, E, Leaver, CJ (1984) Curr. Genet. 8: 537.


  • Ku, HS, Pratt, HK, Spuff, AR, Harris, WM (1968) Plant Physiol. 43: 883.


  • Murray, MG, Thompson, WF (1980) Nucleic Acids Res. 8: 4321.


  • Palmer, D, Shields, CR (1984) Nature (London) 307: 437.


  • Powling, A (1981) Mol. Gen. Genet. 193: 82.


  • Qudtier, F, Vedel, F (1977) Nature (London) 268: 365.


  • Rigby, P, Dieckmann, M, Rhodes, C, Berg, P (1977) J. Mol. Biol. 113: 237.


  • Sparks, Jr, RB, Dale, RMK (1980) Mol. Gen. Genet. 190: 351.


  • Stern, DB, Palmer, JD (1984) PNAS 8: 1946.


  • Synenki, RM, Levings, CS, Shah, DM (1978) Plant Physiol. 61: 460.


  • Vedel, F, Mathieu, C (1982) Anal. Biochem. 127: 1.



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