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