Sample to Insight

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Back to basics: Fundamental concepts and special considerations in gDNA isolationSpeaker: Dr. Marco Polidori

Sample to Insight

Legal Disclaimer

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• 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.

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Agenda

• 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

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Genomic DNA

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

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Sizes and molecular weights of various genomic DNAs

 

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Agenda

• 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

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Sample storage prior to extraction of genomic DNA

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)

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Recommendations for storage of different starting materials

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

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Recommendations for storage of different starting materials

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.

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Agenda

• 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

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Lysis through enzymatic digestion

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

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Handling hard tissues

Mechanical disruption aids in lysis of tougher samples

Disruption using bead millsSamples 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 pestleFor 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.

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Using Fast protocols with new kind of beads

QIAamp Fast DNA Tissue Kit for all soft and hard animal and human tissues

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Systems for mechanical disruption and homogenization

1 sample/run

TissueRuptor

Up to 48 or 192 samples/run

TissueLyser II

Up to 12 samples/run

TissueLyser LT

Human/animal tissuePlant tissue

Human/animal tissuePlant tissueBacteriaYeast

Human/animal tissuePlant tissueBacteriaYeast

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Working with fixed samples

• 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

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Working with fixed samples

• 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

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Agenda

• 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

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DNA extraction technologies

Characteristics of common DNA extraction technologies

  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

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Purification principle of silica (probably…)

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Silica

DNA

Water molecule

Add Ethanol and chaotrop

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Silica column protocol

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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

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gDNA isolation with magnetic beads

Advantages of MagBeads

• Commonly coated with silica residue• Easy to automate • Bead carry-over usually minimal• Requires careful handling if used manually

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Common pitfall: yield versus sample input

Overload is a common issue for DNA isolation using commercial kits

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Agenda

• 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

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Handling & storing DNA

• 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

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Agenda

• 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

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gDNA quality and concentration: UV measurement

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)

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gDNA quality and concentration: UV spectra

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

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gDNA quality and concentration: Fluorophore method

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

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Quality control: gel electrophoresis

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

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Pulse-Field gel electrophoresis (PFGE)

When you need to know the fragmentation of the DNA

194 kb145,5 kb

97 kb

48,5 kb

23,1 kb

9,42 kb6,55 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)

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Agenda

• 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

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 MicroQIAamp DNA Stool MiniQIAamp DNA FFPE TissueQIAamp DNA InvestigatorQIAamp MinElute Media

BloodBone 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)

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DNeasy product line

DNeasy Plant kits

Product name Sample source Kit contents Applications

DNeasy Plant Mini Kit

Plant leaves, root, barkSeeds, fruitFungus<20 mg (dried) or <100 mg (fresh)

QIAshredder MiniBuffer P3DNeasy Mini columnBuffers

Screening transgenic plants

Marker-assisted breeding and mapping

Phylogenetic and biodiversity studies

DNeasy 96 Plant Kit

Buffer P3DNeasy 96 PlateBuffers

DNeasy Plant Maxi Kit

Plant leaves, root, barkSeeds, fruitFungus<100 mg (dried) or <1g (fresh)

QIAshredder MaxiBuffer P3DNeasy Maxi columnBuffers

Specialized protocols: http://www1.qiagen.com/literature; Search for “DNeasy” and select Type “Protocol not contained in handbook”

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Free up you time by automating DNA extraction

Automatable on the QIAcube

Lyse Bind Wash Elute

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Genotyping workflow

Aut

omat

ion

Sam

ple

& A

ssay

Rotor-Gene QPyroMark Q24 & Q96

TissueLyser LT

TissueRuptor

QIAcube EZ1

QIAsymphony

QIAgilityQIAxcel

Detection & analysis

Sample collection & stabilization DNA purification PCR based

genotyping

PAXgene DNA Blood Tubes

Allprotect

RNAlater

QIAampDNeasy

End point PCR kitsType-it product lineEpiTect product line

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gDNA resource center for further information

https://www.qiagen.com/gb/qdm/amp/resourcecenter

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Marco Polidori, Ph.D.Marco.Polidori@qiagen.com

Tel:  +49 2103 29 11441

Questions?

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