Molecular Techniques and Methods

Isolation of Mitochondrial DNA from Green Tissues

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The isolation of mitochondria from green tissues for biochemical studies has been plagued by two problems: mitochondrial fragility and contamination with broken chloroplasts and thylakoid membranes. Etiolated, nongreen differentiated, or nongreen cultured tissues often give better yields of mtDNA than green tissues. Etiolated and nongreen tissues and cultured cells often contain lower concentrations of phenolics or other potentially damaging compounds than green tissues.

In this method, 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 Buffer for mitochondrial isolation contain an osmoticum, EDTA, bovine serum albumin (BSA), 2-mercaptoethanol, and polyvinylpyrrolidone (PVP) which binds phenolics.

  • Yield of mtDNA; 10-20 ug/ 100 g Green Leaves





  • MATERIALS AND SOLUTIONS

    Tissue Grinding Buffer (1 liter)
    0.3 M mannitol ------------------------------------- 54.7 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
    1.0% PVP ----------------------------------------- 10 g
    9 mM 2-mercaptoethanol -------------------------- 625 ul of 14.4 M 2-mercaptoethanol
    Add deionized H2O to make a final volume of ---- 1 liter

    DNase Digestion Buffer (1 liter)
    0.3 M mannitol ------------------------------------ 54.7 g
    50 mM Tris-HCl (pH 8.0) ------------------------- 50 ml of 1 M Tris-HCl
    10 mM EGTA ------------------------------------- 20 ml of 0.5 M EGTA
    5 mM EDTA -------------------------------------- 10 ml of 0.5 M EDTA
    0.2% BSA ----------------------------------------- 2 g
    50 mM MgCl2 ------------------------------------- 50 ml of 1 M MgCl2
    Add deionized H2O to make a final volume of ---- 1 liter


    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


    2.0 M Sucrose Step Gradient Buffer (100 ml)
    2.0 M Sucrose ------------------------------------ 68.5 g
    50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
    10 mM EDTA ------------------------------------ 20 ml of 0.5 M EDTA
    0.1% BSA ---------------------------------------- 0.1 g
    Deionized H2O to make a final volume of -------- 100 ml



    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
    10 mM EDTA ------------------------------------ 20 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
    10 mM EDTA ------------------------------------ 20 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 ------------------------------------ 20.6 g
    50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
    10 mM EDTA ------------------------------------ 20 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

  • All steps should be performed in ice!

    Isolation of Mitochondria from Green Leaves

    1. Slice 100-200 g of fresh green leaves with a knife.

    2. Homogenize tissues in a prechilled Waring blender at high speed for 1 minutes in 5 volumes of Tissue Grinding Buffer.

    3. Filter the homogenate through four layers of cheesecloth and one layer of Miracloth.

    4. Centrifuge the filtrate for 10 minutes at 2,000g and 4oC.

    5. Transfer the supernatant to a new centrifuge tube.

    6. Centrifuge the supernatant for 30 minutes at 10,000g and 4oC.

    7. Resuspend the pellets in 25 ml of DNase Digestion Buffer.

    8. Add 250 ul of DNase (10 mg/ml).

    9. Incubate for 30 minutes on ice,

    10. Add 1 ml of 0.5 M EDTA, and dilute with 300 ml of Gradient Buffer.

    11. Harvest the mitochondria by centrifuging for 15 minutes at 16,000g and 4oC.

    12. Resuspend the pellets with a brush in 6-8 ml of Gradient Buffer.

    13. Layer 3-4 ml of mitochondrial suspension per gradient.
  • Gradients contain 4 ml of 2.0 M Sucrose Step Gradient Buffer, 10 ml of 1.6 M Sucrose Step Gradient Buffer, 10 ml of 1.2 M Sucrose Step Gradient Buffer, and 8 ml of 0.6 M Sucrose Step Gradient Buffer.

    14. Centrifuge for 1 hour at 25,000 rpm.

    15. Collect the two lowest interface bands. The 1.2 M
    ||1.6M interface is predominantly green with an underlying tan layer. The 1.6 M|| 2.0 M interface is predominantly off-white with some green color.

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

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

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

    19. 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

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

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

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

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

    24. 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.

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

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

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

    28. Precipitate DNA at -20oC overnight.

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

    30. 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

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


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


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  • 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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