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Nucleic Acid Extraction: Options, Selection, & Challenges
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Michelle Tabb, PhDVice President, Research & Development
DiaSorin Molecular LLC
Understand the basic principles of nucleic acid extraction
Explore options: from manual to fully automated
Discuss challenges
Future considerations
Objectives
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Extraction: Basic Principles
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Assay Performance
Amplification Chemistry
ExtractionChemistry
Extraction Protocol
Instrument
Extraction Chemistry
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LYSIS
CAPTURE/WASH
ELUTE/PRECIPITATE
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An “Elementary” Example…
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Strawberry DNA Extraction
LYSIS
• Crushing strawberries breaks up cells & releases DNA. • Salt/soap solution frees the DNA from other cell components.• Salt helps DNA strands come together & neutralize the negatively
charged DNA.
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Strawberry DNA Extraction
• Straining clarifies the solution and gets rid of debris
CAPTURE/WASH
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Strawberry DNA Extraction
• DNA is not soluble in alcohol. It forms a white precipitate at the interface of the alcohol and aqueous layer.
ELUTE/PRECIPITATE
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Extraction History
“Old school”
• Friedrich Miescher & Richard Altman – late 1860s
• Density gradient centrifugation – Meselson & Stahl – 1958
• Repeated filtration/precipitation/centrifugation
Boom Method
• Use of silica beads, capable of binding the NA in the presence of a chaotropicsubstance.
• Widespread method for isolating nucleic acids - known as a simple, rapid, and reliable method for the small-scale purification of NA from biological samples
Improvements to Boom
• Protocol optimization for more specific isolation (short ssRNA or long dsDNA)
• Magnetic silica
• Automation
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“Old School” Extraction MethodsSolution-Based
Method Nucleic Acid Purification
Lysis plus Phenol/Chloroform, alcohol preciptiation
DNA
Lysis plus Guanidiniumthiocyanate, sodium acetate,
Phenol/Chloroform
RNA
Alkaline lysis Plasmid DNA
Ethidium bromide/CsCl gradient centrifugation
Plasmid DNA
Ficoll Selective separation of WBC & RBC prior to NA purification
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Extraction History
“Old school”
• Friedrich Miescher & Richard Altman – late 1860s
• Density gradient centrifugation – Meselson & Stahl – 1958
• Repeated filtration/precipitation/centrifugation
Boom Method
• Use of silica beads, capable of binding the NA in the presence of a chaotropicsubstance.
• Widespread method for isolating nucleic acids - known as a simple, rapid, and reliable method for the small-scale purification of NA from biological samples
Improvements to Boom
• Protocol optimization for more specific isolation (short ssRNA or long dsDNA)
• Magnetic silica
• Automation
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Extraction History
“Old school”
• Friedrich Miescher & Richard Altman – late 1860s
• Density gradient centrifugation – Meselson & Stahl – 1958
• Repeated filtration/precipitation/centrifugation
Boom Method
• Use of silica beads, capable of binding the NA in the presence of a chaotropicsubstance.
• Widespread method for isolating nucleic acids - known as a simple, rapid, and reliable method for the small-scale purification of NA from biological samples
Beyond Boom
• Protocol optimization for more specific isolation (short ssRNA or long dsDNA)
• Anion exchange; Magnetic silica
• Manual (Spin columns) and Automation
Extraction: Chaotropic Effect
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Principle: High concentration of salt drives DNA adsorption onto silica, and a low concentration will release the DNA.
Extraction History
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Principle: High concentration of salt drives DNA adsorption onto silica, and a low concentration will release the DNA.
Boom Method
Magnetic Silica
Extraction: Anion Exchange
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Principle: Interaction between positively charged diethylaminoethyl cellulose (DEAE) groupson resin surface and negatively charged phosphates ofthe DNA backbone.
Column-Based ExtractionBasic Principles
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Lyse Bind Wash Elute
Manual versus Automated Options
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Column-Based Extraction Methods(Manual)
Method Nucleic Acid Purification
Silica matrix Glass particles, diatomaceous earth, magnetic silica
Anion-Exchange (DEAE) Thermo Scientific, QIAGEN
From Buckingham & Flaws. 2007. Molecular Diagnostics: Fundamentals, Methods and Clinical Applications
Lyse Bind Wash Elute
Column-Based Extraction (Manual)Invitrogen/Thermo
Column-Based Extraction (Manual)QIAGEN
Column-Based Extraction (Manual)Additional Providers
Promega
Zymo Research
Bioneer
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Automated Extraction Basic Principles
Lyse Bind Wash Elute
Elution time/magnet time
Type of elution buffer
Increase binding time
Extended tip washes
Extended mixing time
Number of washes
Input sample volume vs elution
Amount of lysis buffer
Buffer to sample ratio
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ElutionStep
Lysis Step
WashingSteps
Bead Binding
ConcentrationFactor
Automated Extraction Basic Principles
Incubation time
Elution with/without heat
Specifications NucliSENS® easyMAG® eMAG®
Technology Magnetic Silica-based purification (BOOM Technology)
Input Volume, Elution Volume, &
Sample Type
• 10-1000μl Input• 25-200μl elution• DNA/RNA (Generic-Plasma, Serum, Whole blood, CSF, Sputum, Stool,
Urine, BAL, Swabs, Dry Blood Spot, etc.)
Processing Time 40–60 minutes ~98 minutes
Number of Samples up to 24 up to 48
Dimensions (W × D × H) 39.4 × 25.6 × 20.9 inches Benchtop : 55.9 × 31.5 × 43.7 inchesCabinet : 55.9 × 31.5 × 71.3 inches
Extra Features Reagent Monitoring & LISReagent Monitoring , UV lamp, HEPA
filter, & LIS
bioMérieux Extraction Instruments
SpecificationsMaxwell® CSC
InstrumentMaxwell® RSC Instrument Maxwell® 16 IVD*
Technology Paramagnetic bead-based purification
Input Volume, Elution Volume, &
Sample Type
• 50-300μl input• 50-100μl elution• DNA/RNA-(blood
& FFPE)
• 50-500μl input• 50-100μl elution • DNA/RNA-(Generic-tissue,
saliva, buccal swabs, plants, viral, blood, FFPE, etc.)
• 100-400μl input• 50-100μl elution• DNA/RNA (blood &
viral)
Processing Time 40 – 60 minutes 30 – 60 minutes ~60 minutes
Number of Samples up to 16
Dimensions (W × D × H)
13 × 13.6 × 11.8 inches 12.8 × 17.3 × 12.9
inches
Extra FeaturesUV lamp & Barcode
readerUV Lamp, Barcode reader, &
Integrated QuantitationUV lamp & Barcode
reader
Promega Extraction Instruments
*Available only in Canada & Europe
Specifications QIAcube EZ1 Advanced XL
TechnologySilica-membrane technology
(QIAamp technology)Magnetic Silica-based purification
Input Volume, Elution Volume, &
Sample Type
• 200 μl Input• 20-150 μl elution• DNA/RNA (Plasma, Serum,
Whole blood, CSF, Stool, Urine,Respiratory samples, Cultured cells, Tissue, Forensic specimens)
• 200 - 400 μl Input• 20-150 μl elution• DNA/RNA (Plasma, Serum, Whole
blood, CSF, Stool, Urine,Respiratory samples, Dried swabs)
Processing Time 40 – 60 minutes 25 – 60 minutes
Number of Samples up to 12 up to 14
Dimensions (W × D × H) 25.6 × 24.4 × 22.4 inches 20 × 20 × 22.5 inches
QIAGEN Extraction Instruments
Specifications QIAsymphony
Technology Magnetic Silica-based purification
Input Volume, Elution Volume, &
Sample Type
• 10-1000μl Input• Variable (60 µl – 165 µl)• DNA/RNA (Plasma, Serum, Whole blood, CSF, Sputum, Stool, Urine, BAL,
Respiratory samples, Dry Blood Spot, etc.)
Processing Time 60 – 75 minutes per batch of 24 samples
Number of Samples up to 96 (four batches of 24 samples)
Dimensions (W × D × H) 51.2 × 29.5 × 49.2 inches (SP system)
Extra Features Reagent Monitoring , UV lamp, HEPA filter, & LIS
QIAGEN Extraction Instruments
ASSP
Specifications MagNA Pure LC 2.0 System MagNA Pure 96 System
Technology Magnetic Silica-based purification (BOOM Technology)
Input Volume, Elution Volume, &
Sample Type
• 20 - 1000 μl Input• 50 - 200 μl elution• DNA/RNA - multiple sample
types/protocols
• 50 - 1000 μl Input• 50 - 200 μl elution• DNA/RNA - multiple sample
types/protocols
Processing Time 52 - 180 minutes 50 - 90 minutes
Number of Samples up to 32 up to 96
Dimensions (W × D × H) 40 × 26 × 35 inches 53.9 × 31.5 × 39.4 inches
Roche Extraction Instruments
Selecting Extraction
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• Space
• Budget
• Workflow
• Sample types
• Throughput (manual versus automated)
• Type of assays (i.e. don’t use manual columns for quants)
• Selecting the right chemistry for the type of NA target (amplicon vs virus vs genomic DNA)
• Linked or Integrated
Challenges
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• Extraction reproducibility
• Follow the “Rules of 3”
• Control always in same place on the system?
• Control not extracted on every run?
• How is extraction reproducibility affecting your quant LDT results?
• Extraction instrument calibration
Challenges (continued)
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• Extraction efficiency
• Sample type and kit type
• Are you using a Universal Protocol?
• How good is the recovery?
• How good is the clean up?
Challenges (continued)
32From Buckingham & Flaws. 2007. Molecular Diagnostics: Fundamentals, Methods and Clinical Applications
Challenges (continued)
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From Wilson et l. 2004. J Clin Micro Dec; 42(12): 5913–5916.
Challenges (continued)
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• Concentration factor
• 1X up to ~4X depending upon system and protocol
• Sample input requirements
• Dead volume in automated systems can increase volume requirement
• Contamination
• Robotic system and aerosols
• Manual columns and carry over
Future Considerations
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• Maybe not…for certain applications…and it depends what you consider as “extraction”
• Power of PCR plus minimal sample requirements
• Use of heat +/- centrifugation
Do you really need Extraction?
Do you really need Extraction?
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• Simplexa Direct
• Sample is added directly to Disc
• Combination of chemistry, heat and centrifugation
• Advantages: Nothing lost to extraction efficiency or recovery
• Disadvantages: May not be suitable for all applications i.e. quants
Extraction: Basic Principles
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Assay Performance
Amplification Chemistry
ExtractionChemistry
Extraction Protocol
Instrument
Summary
Extraction can impact downstream applications such as PCR
There are a wide variety of commercially available options for clinical sample extraction
Most current extraction methodologies in use in clinical labs are silica-based
There is not a single solution for every sample type and lab
There are alternatives to traditional extraction
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THANK YOU