Back to Basics: Fundamental Concepts and Special Considerations in gDNA Isolation
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This document provides an overview of genomic DNA (gDNA) isolation. It discusses key considerations for gDNA isolation including sample stabilization, disruption, and storage. Common isolation technologies like silica membrane and magnetic bead kits are described. The document reviews measuring gDNA concentration and quality via UV spectroscopy and gel electrophoresis. It also provides guidance on selecting appropriate QIAGEN gDNA isolation kits based on sample type.
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Back to Basics: Fundamental Concepts and Special Considerations in gDNA Isolation
- 1. Sample to Insight Back to basics: Fundamental concepts and special considerations in gDNA isolation Speaker: Dr. Marco Polidori 1
- 2. Sample to Insight Legal Disclaimer 2 • QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease. • For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.
- 3. Sample to Insight Agenda 3 • gDNA- Introduction • Stabilization of samples • Sample disruption for extraction of genomic DNA • gDNA isolation technologies • Handling and storing gDNA • Measuring concentration and purity of DNA • What method to use
- 4. Sample to Insight Genomic DNA 4 Core questions: How do you break up the cell to access the DNA? • Methods based on cell lysis strongly depend on sample material (e.g. plant vs. blood) • Additional pre-treatments might be necessary (e.g. deparaffinization) How do you get rid of all the other stuff? • Most common biomolecules are relatively easy to remove (proteins, lipids, sugars) • Some metabolites might be co-purified and inhibit your analysis How much DNA can I possibly get out of my sample? • Number of cells available • Some materials contain less living cells or cells with DNA • Size of genome
- 5. Sample to Insight Sizes and molecularweights of various genomic DNAs 5
- 6. Sample to Insight Agenda 6 • gDNA- Introduction • Stabilization of samples • Sample disruption for extraction of genomic DNA • gDNA isolation technologies • Handling and storing gDNA • Measuring concentration and purity of DNA • What method to use
- 7. Sample to Insight Sample storage prior to extraction of genomic DNA 7 The highest DNA yield and quality is achieved by purifying genomic DNA from freshly harvested tissues and cells. If not processing immediately: • Store under conditions that preserve DNA integrity • Genomic DNA yields will decrease if samples, particularly animal samples, are stored at either 2–8°C or –20°C without previous treatment. • Repeated freezing and thawing of frozen samples should be avoided • Use stabilization agents (e.g. AllProtect, RNAlater)
- 8. Sample to Insight Recommendationsfor storage of different starting materials 8 Blood • An anticoagulant should be added, heparin or EDTA can be stored at 2–8°C for a few days or at –20°C or –80°C for a few weeks • Treatment with ACD Solution B (0.48% citric acid, 1.32% sodium citrate, 1.47% glucose; use 1 ml per 6 ml blood) and stored for at least 5 days at 2–8°C or 1 month at –20°C. Other clinical samples • 2–8°C for several hours • Freezing at –20°C or –80°C is recommended for long-term storage • Swabs can be stored dry at room temperature • FFPE storage Animal tissue • Freshly harvested tissue: store at –20°C, –80°C, or in liquid nitrogen • Lysed tissue samples: place in suitable lysis buffer for several months at ambient temperature
- 9. Sample to Insight Recommendationsfor storage of different starting materials 9 Animal, yeast, and bacterial cell cultures • Centrifuge harvested cell cultures, remove the supernatant and then store the cells at –20°C or –80°C. • Alternatively, animal cell nuclei can be prepared and stored at –20°C. Plant tissue • up to 24 hours at 4°C • for durations longer than 24 hours, store at –80°C • Dry storage: Use silica gel, food dehydrators, or lyophilizers etc. Dried samples should be kept in the dark at room temperature under desiccating or hermetic conditions Fungal material • Mycelium should be harvested directly from a culture dish or liquid culture • Harvested samples can be either directly frozen or freeze dried, and stored at – 80°C.
- 10. Sample to Insight Agenda 10 • gDNA- Introduction • Stabilization of samples • Sample disruption for extraction of genomic DNA • gDNA isolation technologies • Handling and storing gDNA • Measuring concentration and purity of DNA • What method to use
- 11. Sample to Insight Lysis through enzymatic digestion 11 Different enzymes help to lyse cells and digest cellular components (Betzel et al. 1993) • Very stable enzymes (e.g. ProtK, Lysozyme) • Usually in the presence of detergents to disrupt membranes… • …and/or chaotropic agents • Additional heat (ProtK: 56°C) • Efficient for soft tissue, cells in culture, blood
- 12. Sample to Insight Handling hard tissues 12 Mechanical disruption aids in lysis of tougher samples Disruption using bead mills Samples are agitated at high speed in the presence of beads. The optimal beads to use are 0.1 mm (mean diameter) glass beads for bacteria, 0.5 mm glass beads for yeast and unicellular animal cells, 3–7 mm stainless steel beads for animal tissues, and 3–7 mm stainless steel or tungsten carbide beads for plant and fungal tissues. Disruption using a mortar and pestle For disruption using a mortar and pestle, freeze the sample immediately in liquid nitrogen and grind to a fine powder under liquid nitrogen. Transfer the suspension (tissue powder and liquid nitrogen) into a liquid-nitrogen–cooled appropriately sized tube and allow the liquid nitrogen to evaporate without allowing the sample to thaw. Add lysis buffer and continue as quickly as possible with the isolation procedure.
- 13. Sample to Insight Using Fast protocols with new kind of beads 13 QIAamp Fast DNA Tissue Kit for all soft and hard animal and human tissues
- 14. Sample to Insight Systems for mechanical disruption and homogenization 14 1 sample/run TissueRuptor Up to 48 or 192 samples/run TissueLyser II Up to 12 samples/run TissueLyser LT Human/animal tissue Plant tissue Human/animal tissue Plant tissue Bacteria Yeast Human/animal tissue Plant tissue Bacteria Yeast
- 15. Sample to Insight Working with fixed samples 15 • Deparaffinization to remove the wax (xylene or deparaffinization solution) • Requires ProtK • Heat treatment to remove cross links FFPE is a special case that requires pre-treatment
- 16. Sample to Insight Working with fixed samples 16 • FFPE is known to contain low frequency artifacts • C>T are the most common transitions found • Can be removed by using UNG Beware of FFPE artefacts Do & Dobrovic; DOI 10.18632/oncotarget.503
- 17. Sample to Insight Agenda 17 • gDNA- Introduction • Stabilization of samples • Sample disruption for extraction of genomic DNA • gDNA isolation technologies • Handling and storing gDNA • Measuring concentration and purity of DNA • What method to use
- 18. Sample to Insight DNA extraction technologies 18 Characteristics of common DNAextractiontechnologies Phenol/Chloroform Anion-exchange Silica-membrane technology Magnetic-particle technology What it is Organic solvent extraction Solid-phase, anion- exchange chromatography Selective adsorption to silica membranes Binding to magnetic silica particles under controlled ionic conditions Procedure Extract with phenol, precipitate and phase separate with choloroform. Alcohol precipitation of DNA Binding: variable salt and pH Elution: variable salt and pH Alcohol precipitation Binding: high salt Elution: low salt Ready-to-use eluate Binding: high salt Elution: low salt Ready-to-use eluate Advantages Efficient lysis, high yields Delivers higher molecular weight DNA (~100 kb) Delivers highly pure DNA for use with all applications. Easy to use, fast, inexpensive. Delivers higher molecular weight DNA if used manually (~200 kb), fast, inexpensive Disadvantages Toxic agents, variable results, carry-over slow difficult to automate Bead carry-over
- 19. Sample to Insight Purification principle of silica (probably…) 19 Silica DNA Water molecule Add Ethanol and chaotrop
- 20. Sample to Insight Silica column protocol 20 1. Bind to silica in high chaotropic salt concentration, low pH 2. Wash away contaminants (chaotropic salt) 3. Wash away chaotropic salts (ethanol) 4. Dry silica membrane 5. Elute in water or low salt buffer, pH 7-9
- 21. Sample to Insight gDNA isolation with magnetic beads 21 Advantages of MagBeads • Commonly coated with silica residue • Easy to automate • Bead carry-over usually minimal • Requires careful handling if used manually
- 22. Sample to Insight Common pitfall: yield versus sample input 22 Overload is a common issue for DNA isolation using commercial kits
- 23. Sample to Insight Agenda 23 • gDNA- Introduction • Stabilization of samples • Sample disruption for extraction of genomic DNA • gDNA isolation technologies • Handling and storing gDNA • Measuring concentration and purity of DNA • What method to use
- 24. Sample to Insight Handling & storing DNA 24 • Avoid introduction of nucleases to DNA solutions • gDNA is relatively fragile and can break, excessive and rough pipetting and vortexing will fragment the DNA • DNA is subject to acid hydrolysis when stored in water, and should therefore be stored in TE buffer • Stored in TE buffer gDNA is stable for decades
- 25. Sample to Insight Agenda 25 • gDNA- Introduction • Stabilization of samples • Sample disruption for extraction of genomic DNA • gDNA isolation technologies • Handling and storing gDNA • Measuring concentration and purity of DNA • What method to use
- 26. Sample to Insight gDNA quality and concentration: UV measurement 26 Light absorption at different wavelengths can tell a few things UV measurement can provide information on yield and purity of gDNA • A230 nm: chaotropic agents, phenol, ethanol • A260 nm: DNA • A280 nm: Protein • A320 nm: Turbidity • 260/280 nm ratio: Should be in range of 1.7-2.0 • 230/260 nm ratio: Should be in range of >1.5 Calculations commonly used • Yield: A260nm – A320nm • Purity: (A260nm – A320nm)/ (A280nm – A320nm) • Purity: (A230nm – A320nm)/ (A260nm – A320nm)
- 27. Sample to Insight gDNA quality and concentration: UV spectra 27 Issues with UV measurement • Prone to overestimating concentrations • 230/260 not indicative of downstream issues! • O.K. as a guide but should not be taken as absolute criteria • Check outlier with additional methods Something is odd (salt, or ethanol carry- over e.g.) Looks how it should look like
- 28. Sample to Insight gDNA quality and concentration: Fluorophore method 28 Fluorophores can be an alternative to UV measurment • Difficult to compare to UV • Much more expensive than UV • Requires dedicated device • If both UV and Qubit give ~same result you have can have good confidence in readout www.thermofisher.com
- 29. Sample to Insight Quality control: gel electrophoresis 29 Standard Gel example • 0.66% TAE gel at 16h, 25V • Most often shows a “smear” • Strong bands at the top of the gel at >20 kb • Can cross check yield readings • Check RNA contamination Lambda / HindIII
- 30. Sample to Insight Pulse-Field gel electrophoresis (PFGE) 30 When you need to know the fragmentation of the DNA 194 kb 145,5 kb 97 kb 48,5 kb 23,1 kb 9,42 kb 6,55 kb 4,36 kb • Higher resolution at larger MWs • Expected to see smear around a peak • Can check MW distribution • Only important for some applications (e.g. de- novo sequencing)
- 31. Sample to Insight Agenda 31 • gDNA- Introduction • Stabilization of samples • Sample disruption for extraction of genomic DNA • gDNA isolation technologies • Handling and storing gDNA • Measuring concentration and purity of DNA • What method to use
- 32. Sample to Insight QIAamp product line QIAamp gDNA purification kits QIAamp DNA Blood • Mini • Midi • Maxi • 96 • BioRobot MDx QIAamp Circulating Nucleic Acid QIAamp DNA Mini QIAamp DNA Micro QIAamp DNA Stool Mini QIAamp DNA FFPE Tissue QIAamp DNA Investigator QIAamp MinElute Media Blood Bone marrow Plasma/Serum Plasma/Serum Tissues Fecal samples Figure sources: Brain, lung, breast: The Web site of the National Cancer Institute (http://www.cancer.gov); Bone: Arne Hendricks(http://flic.kr/p/bXsxwC)
- 33. Sample to Insight DNeasy product line 33 DNeasy Plant kits Product name Sample source Kit contents Applications DNeasy Plant Mini Kit Plant leaves, root, bark Seeds, fruit Fungus <20 mg (dried) or <100 mg (fresh) QIAshredder Mini Buffer P3 DNeasy Mini column Buffers Screening transgenic plants Marker-assisted breeding and mapping Phylogenetic and biodiversity studies DNeasy 96 Plant Kit Buffer P3 DNeasy 96 Plate Buffers DNeasy Plant Maxi Kit Plant leaves, root, bark Seeds, fruit Fungus <100 mg (dried) or <1g (fresh) QIAshredder Maxi Buffer P3 DNeasy Maxi column Buffers Specialized protocols: http://www1.qiagen.com/literature; Search for “DNeasy” and select Type “Protocol not contained in handbook”
- 34. Sample to Insight Free up you time by automating DNA extraction 34 Automatable on the QIAcube Lyse Bind Wash Elute
- 35. Sample to Insight Genotyping workflow AutomationSample&Assay Rotor-Gene Q PyroMark Q24 & Q96 TissueLyser LT TissueRuptor QIAcube EZ1 QIAsymphony QIAgility QIAxcel Detection & analysis Sample collection & stabilization DNA purification PCR based genotyping PAXgene DNA Blood Tubes Allprotect RNAlater QIAamp DNeasy End point PCR kits Type-it product line EpiTect product line
- 36. Sample to Insight gDNA resource center for further information https://www.qiagen.com/gb/qdm/amp/resourcecenter
- 37. Sample to Insight Marco Polidori, Ph.D. Marco.Polidori@qiagen.com Tel: +49 2103 29 11441 Questions? Thank you for attending Contact QIAGEN Call: 1-800-426-8157 qiawebinars@qiagen.com