Feb 01, 2018
Isolation of high-quality, highly enriched mitochondrial DNA from mouse tissues
- Marita A. Isokallio1,
- James B. Stewart1
- 1Max Planck Institute for Biology of Ageing
Protocol Citation: Marita A. Isokallio, James B. Stewart 2018. Isolation of high-quality, highly enriched mitochondrial DNA from mouse tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.mycc7sw
Manuscript citation:
Johanna H K Kauppila, Nina A Bonekamp, Arnaud Mourier, Marita A Isokallio, Alexandra Just, Timo E S Kauppila, James B Stewart, Nils-Göran Larsson; Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice, Nucleic Acids Research, , gky456, https://doi.org/10.1093/nar/gky456
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 01, 2018
Last Modified: April 19, 2018
Protocol Integer ID: 9956
Keywords: mitochondrial DNA, mtDNA, mtDNA isolation, mitochondrial genetics
Abstract
One remarkable issue in mitochondrial DNA sequencing and variant detection is heavy nuclear DNA contamination persistent throughout the commonly used mitochondria enrichment protocols including differential and gradient centrifugations [1,2]. The chromosomes contain nuclear mitochondrial sequences [3], which might cause false positive or negative results in variant detection. This problem is often overcome by enrichment of the mtDNA by long-range PCR [4], however, such methods are prone to early-cycle PCR errors. Here, we improved existing mtDNA isolation protocols (e.g. Kennedy et al. (2013) [11]) and describe a detailed step-by-step guide to obtain high-quality, highly enriched mtDNA suitable for sequencing and low-frequency variant detection as well as for other sensitive applications.
Guidelines
INTRODUCTION
Although a cell harbors several thousands of mtDNA molecules, only <1 % of a total genomic DNA extraction is mtDNA. Thus, experiments focusing on mtDNA analysis often include an mtDNA enrichment step. Traditionally mitochondria are enriched from the cellular debris with differential or gradient centrifugation methods [1,2] or by amplification-based enrichment of the mtDNA [4,5]. The former methods will only partially enrich mtDNA, but samples still contain high nuclear DNA contamination levels [6]. Amplification-based methods suffer from errors caused by DNA polymerases or unintended amplification of nuclear sequences of mitochondrial origin (NuMTs) [7].
In the mouse genome, 172 chunks ranging from 33 bp to 4.7 kbp in length with 66–100 % identity were identified [8]. Others estimated that >95 % of the mouse mtDNA genome is present in the nuclear chromosomes [9]. NuMTs may be present or absent in different individuals and mtDNA is constantly being transferred to the nucleus, thus, NuMT content varies between individuals [7,8]. All these characteristics of NuMTs complicate the design of primers for mtDNA enrichment, and thus, might affect amplification- or blotting-based mtDNA studies [4,10]. Furthermore, ever increasing deep sequencing approaches to detect mtDNA mutations are especially prone for false results if a large amount of nuclear DNA is present in the sample. As NuMTs may be fully homologous to true mtDNA sequences, results would be biased towards wild-type reads [8]. On the other hand, polymorphic NuMTs would be observed as false-positive mutations which are impossible to distinguish from true mtDNA mutations [8].
Computational approaches [10] or fast plasmid preparations in combination with PCR-amplification [12] have been suggested as solutions to avoid NuMT reads in mtDNA mutation detection or to highly enrich mtDNA. Computational approaches, however, are very challenging to implement, whereas fast DNA extraction procedures and amplification step may decrease the quality of the DNA and expose it to damage. DNA damage may also appear as false-positive mutations in sequencing applications, as DNA polymerase, for example, might bypass the damaged site by incorporating a wrong nucleotide or damage might increase the polymerase jumping increasing the amount of chimeric DNA molecules [14]. Furthermore, artefacts caused by oxidative damage to the DNA, often observed as G>T/C>A mutations, are a remarkable issue in reliable variant detection [15,16]. Hence, we reasoned that mtDNA analyses, especially variant detection by deep sequencing, should begin with high-quality, highly enriched mtDNA sample. Here, we describe a step-by-step protocol to extract mtDNA from mouse tissues suitable for highly-sensitive mtDNA experiments. The extremely high enrichment of mtDNA from nuclear DNA contamination is based on gentle tissue homogenization, extensive DNase I treatment of the isolated, intact mitochondria (modified from the protocol described by Kennedy et al. [2013] [11]) and DNA extraction by chloroform:isoamyl alcohol and ethanol precipitation.
References
1. Frezza, C. et al. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroplasts. Nat. Protoc. 2, 287-295 (2007).
2. Wieckowski, M.R. et al. Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nat. Protoc. 4, 1582-1590 (2008).
3. Hazkani-Covo, E. et al. Molecular poltergeist: Mitochondrial DNA copies (numts) in sequenced nuclear genomes. PLoS Genet. 6, e1000834 (2010)
4. Payne, B.A.I. et al. Deep Resequencing of Mitochondrial DNA. Methods Mol. Biol. 1264, 59-66 (2015).
5. Ni, T. et al. MitoRCA-seq reveals unbalanced cytocine to thymine transition in Polg mutant mice. Sci. Reports 5, 12049 (2015).
6. Ameur, A. et al. Ultra-deep sequencing of mouse mitochondrial DNA: mutational patterns and their origins. PLoS Genet. 7, e1002028 (2011).
7. Hazkani-Covo, E. et al. Molecular poltergeists: mitochondrial DNA copies (numts) in sequenced nuclear genomes. PLoS Genet. 6, e1000834 (2010).
8. Calabrese, F.M., et al. Primates and mouse NumtS in the UCSC Genome Browser.
BMC Bioinformatics 13, S15 (2012).
9. Malik, A.N. et al. Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences. Mitochondrion 29, 59–64 (2016).
10. Li, M. et al. Fidelity of capture-enrichment for mtDNA genome sequencing: Influence of NUMTs. Nucleic Acids Res. 40, e137 (2012).
11. Kennedy, S.R. et al. Ultra-Sensitive Sequencing Reveals an Age-Related Increase in Somatic Mitochondrial Mutations That Are Inconsistent with Oxidative Damage. PLoS Genet. 9, e1003794 (2013).
12. Samuels, D.C. et al. Finding the lost treasures in exome sequencing data. Trends Genet. 29, 593–599 (2013).
13. Quispe-tintaya, W. et al. Fast mitochondrial DNA isolation from mammalian cells for next-generation sequencing. Biotechniques 55, 133–136 (2015).
14. Eckert, K.A. & Kunkel, T.A. DNA polymerase fidelity and the polymerase chain reaction. PCR Methods Appl. 1, 17–24 (1991).
15. Costello, M. et al.. Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation. Nucleic Acids Res. 41, 1–12 (2013).
16. Chen, L. et al. DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification. Science 355, 752–756 (2017).
Acknowledgements
This work was supported by Max Planck Society.
Materials
MATERIALS
37-% Hydrochloric acidMerck MilliporeCatalog #1.00317.1000
Acetic acid, glacialMerck MilliporeCatalog #1.01830.2500
Bovine serum albumin, essentially fatty-acid freeSigma AldrichCatalog #A6003
Calcium chloride Merck MilliporeCatalog #1.02378.0500
Chloroform:isoamylalcohol 24:1AmrescoCatalog #X205
Deoxyribonuclease I from bovine pancreas, type IVSigma AldrichCatalog #D5025
dNTP Set, illustra Solution, 100 mM each dNTPGe HealthcareCatalog #28-4065-52
Elution buffer (5 mM Tris/HCl, pH 8.5) Macherey and NagelCatalog #740917.1
Ethanol, absolute 99.8%Catalog #10342652
Ethidium bromide 10 mg/mlSigma AldrichCatalog #E1510
Ethylenediaminetetraacetic acid disodium salt dihydrateSigma AldrichCatalog #ED2SS
Ethylene-bis(oxyethylenenitrilo)tetraacetic acidSigma AldrichCatalog #E3889
GeneRuler 1 kb DNA LadderThermo Fisher ScientificCatalog #SM0311
GeneRuler 100 bp DNA Ladder Thermo Fisher ScientificCatalog #SM0241
GoTaq® Flexi DNA PolymerasePromegaCatalog #M8291
Magnesium chloride hexahydrateSigma AldrichCatalog #M2670
Lambda DNA/HindIII MarkerThermo Fisher ScientificCatalog #SM0101
Potassium acetateMerck MilliporeCatalog #1.04820.1000
Proteinase K, recombinant, PCR gradeSigma AldrichCatalog #3115836001
Qubit® dsDNA HS Assay kitThermo Fisher ScientificCatalog #Q32854
RNase AMacherey and NagelCatalog #740505
Sodium acetateMerck MilliporeCatalog #1.06268.1000
Sodium dodecyl sulfate, pelletsCarl RothCatalog #CN30.2
Sodium hydroxideMerck MilliporeCatalog #1.06498.5000
SucroseSigma AldrichCatalog #S0389
Tris(hydroxymethyl)aminomethaneMerck MilliporeCatalog #1.08382.2500
Ultrapure(TM) AgaroseThermo Fisher ScientificCatalog #16500100
Ultrapure(TM) TBE buffer, 10xThermo Fisher ScientificCatalog #15581-0280
Primer: PolgA_F CTTCGGAAGAGCAGTCGGGTGSigma Aldrich
Primer: PolgA_R GGGCTGCAAAGACTCCGAAGGSigma Aldrich
Before start
The protocol here is presented for a single tissue sample, but it can be scaled up for simultaneous preparation of several samples. However, significantly prolonged incubation times at any step due to increased amount of samples should be avoided.
The protocol has been mainly optimized for soft tissues, such as liver and brain (as in [11]). However, it also works for heart, although more extensive yet gentle homogenization is required. The mtDNA yield from heart is also much lower than from the bigger tissues and nDNA contamination levels are often higher.
REAGENT SETUP
CRITICAL: Reagent storage is at room temperature unless mentioned otherwise.
STOCK SOLUTIONS
• Acetic acid, 2 M Combine 11.48 ml glacial acetic acid with 88.52 ml distilled H2O to obtain 100 ml 2 M acetic acid solution.
• DNase I, 5 mg/ml in 5 mM CaCl2 Dissolve DNase I according to the weight into suitable volume of 5 mM CaCl2 (diluted from 1 M CaCl2 stock solution, e.g. 7.5 µl 1 M CaCl2 in 1.5 ml distilled H2O) to obtain 5 mg/ml DNase I solution. Dispense into aliquots and store at -20 °C.
CRITICAL: DNase I activity might decrease if the storage period exceeds several months.
• dNTPs, 1.25 mM each Combine 250 µl of each dNTP (100 mM stocks) and add 19 ml distilled H2O to obtain a solution containing 1.25 mM each dNTP. Dispense into aliquots and store at -20 °C.
• EDTA, 0.5 M, pH 8.0 Dissolve 18.1 g EDTA into 80 ml distilled H2O. Adjust the pH to 8.0 with NaOH and bring the volume to 100 ml with distilled H2O.
• EtOH, 70-% Measure 35 ml of absolute EtOH and 15 ml distilled H2O separately and combine to obtain ca. 48 ml of 70-% EtOH.
CRITICAL: 70-% EtOH is hygroscopic i.e. it will evaporate and absorb water over time, which will lower the concentration. Thus, always use rather freshly prepared 70-% EtOH solution.
• NaOH, 15 M Dissolve 600 g NaOH into 600 ml distilled H2O. Bring the volume to 1 liter with distilled H2O.
CAUTION! The reaction is exothermic. The solution can be kept on ice during preparation.
• Potassium acetate, 6 M Dissolve 58.92 g potassium acetate into 40 ml distilled H2O. Adjust the pH to 7.5 with 2 M acetic acid. Bring the volume to 100 ml with H2O.
CRITICAL: Long-term storage is recommended at -20 °C.
• Proteinase K, 10 mg/ml Dissolve 100 mg Proteinase K into 10 ml of distilled H2O. Dispense into aliquots and store at -20 °C.
• RNase A (free of DNase), 9.09 mg/ml Dissolve RNase A at a concentration of 10 mg/ml into 10 mM sodium acetate pH 5.2 (e.g. dissolve 100 mg of RNase A into 1 ml of distilled H2O, and dilute 100 µl of that RNase A solution with 890 µl of distilled H2O and 10 µl of 1 M sodium acetate). Incubate the aliquot tubes in a heat block at 100 °C for 15 min. Allow the tubes to slowly cool down to room temperature. Adjust the pH by adding 100 µl (i.e. 0.1 volumes) of 1 M Tris (pH 7.4) to obtain 9.09 mg/ml RNase A solution. Dispense into aliquots and store at -20 °C.
CRITICAL: RNase A precipitates if the concentrated solution is boiled at neutral pH.
• SDS, 10-% Dissolve 10 g SDS into 80 ml distilled H2O. Bring the volume to 100 ml with distilled H2O.
• Sodium acetate, 1 M, pH 5.2 Dissolve 24.61 g sodium acetate into 50 ml distilled H2O. Adjust the pH to 5.2 with glacial acetic acid. Allow the solution to cool down to room temperature and adjust the pH again to 5.2 with glacial acetic acid. Bring the volume to 100 ml with distilled H2O.
• Tris, 1 M, pH 7.4 Dissolve 12.11 g Tris into 80 ml distilled H2O. Adjust the pH to 7.4 with HCl. Allow the solution to cool down to room temperature and adjust the pH again with HCl. Bring the volume to 100 ml with distilled H2O.
• Tris, 1 M, pH 8.0 Prepare as Tris, 1 M, pH 7.4, but adjust the pH to 8.0.
BUFFERS
• Lysis buffer: 20 mM Tris, 150 mM NaCl, 20 mM EDTA, 1 % SDS, pH 8.75 Dissolve 0.877 g NaCl into 60 ml distilled H2O. Add 2 ml Tris (1 M, pH 8.0), 4 ml EDTA (0.5 M, pH 8.0) and 10 ml SDS (10-%). Adjust the pH to 8.75 with NaOH at room temperature. Bring the volume to 100 ml with distilled H2O.
CRITICAL: High concentrations of NaCl and SDS precipitate.
CRITICAL: Tris pH is temperature dependent: pH 8.75 at room temperature corresponds to pH 7.8 at the usage temperature of 56 °C.
• Mitochondria isolation buffer (MIB), 2x concentrate: 640 mM sucrose, 40 mM Tris, 2 mM EGTA, pH 7.2 Dissolve 219 g sucrose, 4.58 g Tris, 0.76 g EGTA into 950 ml distilled H2O. Adjust the pH to 7.2 with HCl and bring the solution to 1 liter with distilled H2O. Aliquot and store the 2x concentrate MIB at -20 ℃ until use.
CRITICAL: Tris pH is temperature dependent: pH 7.2 at room temperature corresponds to pH ca. 7.8 at the usage temperature of 0-4 ℃.
CRITICAL: MIB 2x concentrate solution can be stored frozen for very long periods of time (>12 months).
• Mitochondria isolation buffer (MIB02): 320 mM sucrose, 20 mM Tris, 1 mM EGTA, 0.2 % BSA, pH 7.2 Thaw 2x concentrate MIB and dilute it with equal volume of distilled H2O to obtain 1x MIB solution. Add 0.2 % (w/v) BSA to obtain MIB02 solution.
CRITICAL: Store thawed MIB at 4 ℃ only short periods of time (1-2 days).
CRITICAL: BSA-containing MIB solution should always be prepared freshly on the day of usage and stored on ice or at 4 ℃.
• Mitochondria isolation buffer (MIB1): 320 mM sucrose, 20 mM Tris, 1 mM EGTA, 1 % BSA, pH 7.2 Thaw 2x concentrate MIB and dilute it with equal volume of distilled H2O to obtain 1x MIB solution. Add 1 % (w/v) BSA to obtain MIB1 solution.
CRITICAL: Store thawed MIB at 4 ℃ only short periods of time (1-2 days).
CRITICAL: BSA-containing MIB solution should always be prepared freshly on the day of usage and stored on ice or at 4 ℃.
• Mito-DNase buffer base: 300 mM sucrose, 10 mM MgCl~2~, 20 mM Tris, pH 7.5 Dissolve 10.27 g sucrose, 0.203 g MgCl2 and 0.243 g Tris into 90 ml distilled H2O. Adjust the pH to 7.5 with HCl and bring the solution to 100 ml with distilled H2O. Aliquot and store the Mito-DNase buffer base at -20 ℃ until use.
CRITICAL: Tris pH is temperature dependent: pH 7.5 at room temperature corresponds to pH ca. 7.2 and 8.1 at the usage temperatures of 37 and 0-4 ℃, respectively.
• Mito-DNase buffer: Mito-DNase buffer base, 0.15 % (w/v) BSA, 0.03 mg/ml DNase I, 0.02 mg/ml RNase A Thaw Mito-DNase buffer base and add 0.15 % (w/v) BSA. Just before use, add 0.03 and 0.02 mg/ml DNase I and RNase A, respectively.
CRITICAL: BSA-containing Mito-DNase solution should always be prepared freshly on the day of usage and stored on ice or at 4 ℃.
EQUIPMENT
• 1.5-ml microcentrifuge tubes
• 250-µl low-bind, wide-orifice pipette tips (VWR, cat. no. 613-0370)
• 50-ml polypropylene Falcon tubes
• Gel electrophoresis system (e.g. Bio-Rad Sub-Cell(TM) GT Cell)
• Gel imaging system (e.g. Syngene U:Genius)
• Heat block for 1.5 ml microcentrifuge tubes (e.g. Grant QB-H2)
• Motor-driven glass/Teflon Potter Elvehjem homogenizer (e.g. Sartorius Potter S)
• PCR tubes (e.g. VWR® 8-well tube strips with bubble cap)
• PCR thermocycler (e.g. The Applied Biosystems® Veriti® 96-Well Thermal Cycler)
• Qubit® Assay tubes (Thermo Fisher Scientific, cat.no. Q32856)
• Qubit® 3.0 Fluorometer (Thermo Fisher Scientific, cat.no. Q33216)
• Refrigerated centrifuge for 1.5 ml microcentrifuge tubes (e.g. Eppendorf 5417R)
• Refrigerated centrifuge for 50-ml Falcon tubes, swing- and fixed rotors (e.g. Eppendorf 5804R)
Tissue collection and homogenization (1-1.5 hour)
Tissue collection and homogenization (1-1.5 hour)
Sacrifice a mouse and collect 500-800 mg of liver tissue or a full brain (ca. 430 mg). Rinse the tissue in ice cold PBS. Store the tissue in a 50-ml Falcon tube in 5-10 ml ice cold PBS until homogenization.
Note
Use only freshly collected tissue for mitochondria isolation.
Homogenize the tissue with a teflon pestle by 5 strokes with 200 rpm. (Experimental: Homogenization of heart tissue requires harsher conditions.)
Note
Avoid breakage of the nucleus or mitochondria by too rigorous homogenization.
Remove PBS and transfer the tissue into glass homogenizer tube with 15 ml ice cold MIB1.
Transfer the homogenate back to the 50-ml Falcon tube and add 15 ml ice cold MIB1
Mitochondria isolation (~1 hour)
Mitochondria isolation (~1 hour)
Remove cell debris by centrifugation at 800 g 4 ℃ for 10 min.
4 °C 800g
00:10:00
Pour the supernatant into a clean 50-ml Falcon tube and repeat the centrifugation.
Note
In case a fat layer is formed on top of the liquid, dip a pipet tip gently into the solution and remove as much fat as possible.
00:10:00
4 °C 800g
Pour the supernatant carefully into a clean 50-ml Falcon tube.
Note
Do not disturb the pellet.
Collect mitochondria by centrifugation at 8,500 g 4 ℃ for 10 min.
00:10:00
4 °C 8500g
iscard supernatant by pouring and carefully pipet out all leftover liquid. Continue to the next step immediately.
Nuclear DNA removal (~3 hours)
Nuclear DNA removal (~3 hours)
Add 0.03 mg/ml DNase I and 0.02 mg/ml RNase A into ice cold Mito-DNase buffer.
Dissolve mitochondria pellet carefully into 600 µl Mito-DNase buffer by pipetting up and down. Keep the tubes on ice.
Note
Use wide-orifice pipet tips when pipetting mitochondria-containing solutions.
Note
Harsh pipetting will break mitochondria and decrease the overall yield, but thorough dissolving is required for DNase I to access and digest any nDNA leftovers increasing the purity of the final mtDNA sample.
Divide dissolved mitochondria solution into subfractions: the number of fractions depend on the input tissue amount such that each fraction represents ca. 100-150 mg tissue in 600 µl Mito-DNase buffer. For example, for a full brain tissue (ca. 430 mg) add 2x 600 µl Mito-DNase buffer (final volume 1.8 ml), mix well by pipetting and divide the solution into three 1.5-ml microcentrifuge tubes.
Note
Use wide-orifice pipet tips when pipetting mitochondria-containing solutions.
Incubate the tubes at 37 ℃ for 1-1.5 hours.
01:00:00
37 °C
Collect mitochondria by centrifugation at 13,000 g 4 ℃ for 30 min.
00:30:00 13000g
Remove supernatant thoroughly.
Wash out the leftovers of Mito-DNase buffer by carefully re-suspending the mitochondria pellet into 500 µl MIB02, and re-pelleting by centrifugation at 13,000 g 4 ℃ for 15 min.
00:15:00
4 °C 13000g
Repeat the previous wash step.
00:15:00
4 °C 13000g
Remove the supernatant, spin down the leftover liquid and pipet out any traces of liquid.
Freeze the pellet in liquid nitrogen and store at -80 ℃ until DNA extraction.
Note
Mitochondria pellets can be stored at -80 ℃ at least a few hours. Longer storage times (weeks/months) is not expected to affect the mtDNA extraction, but has not been tested during this protocol optimization.
DNA extraction (~3-20 hours)
DNA extraction (~3-20 hours)
Add 0.02 mg/ml Proteinase K and 0.02 mg/ml RNase A into lysis buffer preheated to 56 ℃.
56 °C
Dissolve frozen mitochondria pellets into 400 µl lysis buffer by pipetting up and down vigorously.
Incubate the tubes at 56 ℃ overnight (ca. 14-16 hours).
16:00:00
56 °C
Note
Shorter lysis time can be used, even 1-hour lysis should be sufficient, but has not been tested during this protocol optimization.
Let the solution cool down to room temperature.
Add 100 µl potassium acetate.
Note
White precipitate forms when potassium reacts with SDS.
Add 500 µl of chloroform:isoamylalcohol (24:1) and shake the tubes for 20 s.
00:00:20
Separate the phases by centrifugation at 16,000 g at room temperature for 10 min.
00:10:00
22 °C 16000g
Transfer the upper aqueous phase carefully to a clean 1.5-ml microcentrifuge tube.
Add 100 or 200 µg of RNase A (i.e. 11 or 22 µl for brain or liver sample, respectively).
Note
Complete removal of RNase A might require additional chloroform:isoamylalchol purification step instead of EtOH precipitation alone. However, additional purification step may decrease the final yield and is not necessarily required for a successful Illumina sequencing.
Incubate the tubes at 37 ℃ for 45-60 min.
00:45:00
37 °C
Add 1 ml (2 volumes) ice cold absolute EtOH and invert the tubes gently for 5 times.
Note
If, for example, smaller amount of tissue than recommended in this protocol is used and low concentration of DNA expected, glycogen can be used as a carrier to increase the DNA yield in EtOH precipitation. Even glycogen with a dye (e.g. Glycoblue) is compatible with Illumina sequencing.
Incubate the tubes at -80 ℃ at least for 30 min.
03:00:00
-80 °C
Note
Incubation can be even 2-3 hours. Alternatively, prolonged (e.g. overnight) incubation at -20 ℃ is possible.
Pellet DNA at 16,000 g at room temperature for 15 min.
00:15:00
22 °C 16000g
Remove supernatant by pipetting and wash DNA with 500 µl 70 % EtOH. Pellet DNA by centrifugation at 16,000 g at room temperature for 5 min.
00:05:00
22 °C 16000g
Repeat the wash step in order to increase the purity of the DNA.
00:05:00
22 °C 16000g
Note
Do not disturb the pellet.
After the last wash, spin down the leftover EtOH and remove all traces by pipetting.
Air-dry the pellet briefly (1-2 min) until all trace ethanol has evaporated.
Note
Over-drying the DNA might lead to a non-dissolving pellet or damage the DNA.
Drying the pellet with heat is not recommended as higher temperatures may also damage the DNA.
Dissolve the DNA pellet by adding 18-30 µl elution buffer AE (5 mM Tris/HCl, pH 8.5) depending on the DNA pellet size and required final concentration. Incubate the tubes at room temperature overnight.
16:00:00
Note
Dissolving the pellet with heat is not recommended as higher temperatures may damage the DNA.
Note
DNA is less stable in H~2~O and pH of H~2~O might not be optimal for DNA storage, whereas EDTA of TE buffer might not be suitable for downstream applications such as Illumina sequencing.
Note
High yield of mtDNA might appear as a brown pellet and brown color might be visible also in the dissolved DNA solution. This affects spectrophotometric DNA concentration measurement, thus, fluorometric method should be used in order to have more reliable concentration measurement.
Note
If higher concentration of DNA is required, it is advisable to use 5 mM Tris buffer. Doubling the concentration of DNA can then be obtained by evaporating half of the sample volume, giving a final concentration of 10 mM Tris which is suitable for most downstream applications.
Short-term storage is recommended at 4 ℃, long-term storage at -20 ℃.
Note
Multiple freeze-thaw cycles should be avoided.
Note
Continue to the next step earliest on the following day of dissolving the DNA, or stop the protocol here and store the DNA at 4 or -20 ℃ according to the length of the pause. However, it is recommended to continue with the quality control measurements and combination of the final samples before long-term storage of the DNA sample at -20 ℃ in order to avoid unnecessary freeze-thaw cycles.
DNA quality control (~3-4 hours)
DNA quality control (~3-4 hours)
Measure the concentration of 1 µl sample with Qubit HS kit twice and take the average as the sample concentration. If the sample is too concentrated, remeasure with suitable dilution.
Test for nDNA contamination by performing 20-µl PCR reaction with primers designed for an nDNA-encoded target (here, we use primers specific for PolgA (expected product size 520 bp).
Master mix for a single 20-µl PCR reaction:
| µl | Reagent | Final concentration | |
| 8.9 | H2O | adjust according to the sample volume | |
| 4.0 | 5x Green GoTaq reaction buffer | 1x | |
| 3.2 | dNTPs (1.25 mM each) | 200 µM | |
| 1.2 | MgCl2 (25 mM) | 1.5 mM | |
| 0.8 | PolgA_F (10 µM) | 0.4 µM | |
| 0.8 | PolgA_R (10 µM) | 0.4 µM | |
| 0.1 | GoTaq DNA polymerase (5u/µl) | 0.5 units | |
| 1.0 | mtDNA sample | see note |
PCR programme:
94 C - 60 s
---------------
30 cycles of:
94 C - 30 s
58 C - 30 s
72 C - 45 s
---------------
72 C - 3 min
8 C - Inf
Note
Generally, 1 µl of the sample is suitable for the PCR, however, if sample concentrations are highly variable between samples, it is advisable to normalize the input DNA amounts.
Note
Use genomic DNA, such as a quick tail extract, as a positive control.
Note
For more accurate quality control, quantitative PCR is recommended as described by Kennedy et al. (2013) [11]
Note
Heavy RNA contamination inhibits the PCR reaction and might give false-negative result.
Load the 20-µl PCR reaction fully on 1-% agarose gel in 0.5x TBE buffer containing 0.5 µg/ml EtBr. Only load 5 µl or less of the positive control sample.
Note
No or only very slightly visible DNA band should be observed on the gel (see Figure 1A). nDNA contaminated fractions should not be used for further analysis. The isolation protocol should be optimized, often starting from the homogenization step, in order to reduce the nDNA contamination.
Note
Mitochondrial DNA enrichment can be verified by PCR reaction with primers specific for a mtDNA-encoded gene.
Combine fractions from the same tissue which show no or extremely minor nDNA contamination and re-measure the final sample concentration with Qubit HS. Dispense the sample into aliquots and store at -20 ℃.
Note
If higher (maximum double) concentration is needed for the downstream application, concentrate the sample by vacuum at 45 ℃.
Test the quality of the mtDNA sample by running 100 ng (according to the Qubit HS concentration measurement) on 1-% agarose gel.
Note
Circular mtDNA shows multiple bands on the gel according to different forms: supercoiled plasmid, relaxed plasmid and/or linear DNA. There should not be strong smears.
Note
Especially in a short gel run (ca. 30 min), RNA contamination can be seen as two distinct low-molecular weight bands, or more significant RNA contamination might also appear as a huge blob (see Figure 1C, lanes 1 and 4).
Expected result
Expected result
From liver tissue, total yield of 2-3 µg of good quality mtDNA should be obtained: nuclear DNA specific PCR should not give a visible product on the gel and mtDNA should show distinct bands on the gel.
From brain tissue, total yield of 200-500 ng of highly enriched mtDNA should be obtained. Often slight nDNA contamination can be detected by PCR, but mtDNA should show distinct bands on the gel.
See Figure 1 for the examples of PCR results and gel runs.