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APPROACHES TO NUCLEIC ACID EXTRACTION
Richard L. Hodinka, Ph.D. University of South Carolina School of Medicine Greenville
Greenville Health System, Greenville, SC hodinka@greenvillemed.sc.edu
Objectives of Talk
Describe the various technologies and advances in nucleic acid purification
Discuss the principles involved in extraction of nucleic acids
Compare and contrast some of the available molecular instruments for nucleic acid extraction
Ingredients Needed for Success with Molecular Assays
Specimens
NA Isolation
Reagents
Facilities
Amplification
Detection
Quality Control
Quality Assurance
Result Analysis &
Interpretation
Specimen Collection & Handling
Specimen collection and handling can have a significant effect on final test results
Integrity of target nucleic acid sequence must be maintained
Clearly defined criteria should be established for proper specimen collection, labeling, preservation, transportation, and storage of specimens
Establish educational programs
Have a firm (but fair) rejection policy
Develop guidelines for specimen processing and carefully manage procedures
Practical Rules of Thumb
As a general rule, submit specimens to laboratory as quickly as possible after collection Keep cold (2-8oC) for short-term transit and storage;
avoid extend times at RT
Freeze to at least -70oC or below for long-term transit or storage following initial processing
Once received by laboratory, processing and storage of specimens for testing should proceed in a timely manner
Store samples in single-use aliquots
Specimen material should not be returned to original container after use for molecular testing
Avoid multiple freeze/thaws; should not exceed three freeze/thaw cycles
Nucleic Acid Extraction
Refers to the release of target nucleic acid from its surrounding material
Key fundamental step to subsequent downstream molecular analysis
Can isolate DNA, RNA or Both
Quality/adequacy of target nucleic acid
Reduction or inactivation of any inhibitory/interfering substances
Nucleic Acid Extraction
What’s available? What’s best for me?
Do I use manual or automated systems?
Do I have the needed resources and space?
If automated, which platform should I select?
What number/types of samples will be run?
Do I need DNA, RNA, or both?
What about recovery and quality of purified NA?
What about interfering substances and cross contamination?
How can I best standardize the approach?
Basic Steps of Nucleic Acid Isolation
Pre-processing of samples, if needed
e.g., tissue, stools, whole blood
Lysis of microorganisms to release
nucleic acids
Extraction to remove unwanted cellular
components
Purification of nucleic acids
Bead Beating Cell/Tissue Disruption
Physical disruption before extraction
Grinding Beads
Shaking Homogenizers
Traditional NA Extraction Methods
Treating sample with heat, alkali, sonication
Use of detergents and enzymes
Extraction with organic solvents and precipitation in presence of salts and cold alcohols
Organic Extraction Methods
Accomplished using an organic mixture of phenol-chloroform
Nucleic acid separates into an aqueous phase, lipids in the organic phase, and proteins at the interphase
Keeping the pH at 7.0-8.0 and adding isoamyl alcohol would help keep RNA in aqueous phase
openwetware.org/wiki/phenol/chloroform_extraction
Organic Extraction Methods
Require considerable
labor, time, costs
Multiple
manipulations
Technical burden of
processing multiple
specimens
Difficult to train and
maintain skilled staff
Low Yields
Recovery of partially
degraded material
Co-purification of
contaminants
Variation within and
between extraction
runs
Inorganic Extraction Methods
Developed over the years to replace
organic extractions
Samples are lysed
Proteins and other contaminants are
selectively precipitated
The nucleic acids are then precipitated,
washed, and resuspended
Inorganic Extraction Methods
Many commercial kits available
Complete kits for purification of DNA, RNA, or total nucleic acids from biological materials
Selected commercial vendors Epicentre MasterPure
Gentra Systems Puregene
Orca Research IsoQuick
BioMerieux NucliSens
Roche Molecular Isolation Kits
Schleichar & Schuell IsoCode Stix
Mo Bio Molecular Isolation Kits
Advances in NA Isolation
Solid support methods
Significant improvement in speed,
efficiency and standardization of the
process
Increased availability and development
of commercial reagents and systems
Automation
Solid Support Methods
More rapid and user-friendly systems
Use solid matricies (silica or glass
particles) to bind and purify nucleic acids
in presence of chaotropic agents
Many commercial systems based on this
technology
Produce nucleic acid preparations of
high quality and high purity
Selected Commercial Spin Columns
Qiagen QIAmp, RNeasy
Clontech NucleoSpin
Amresco Cyclo-Prep
Gentra Systems Generation Capture
Stratagene Strata Prep
Roche Molecular High Pure
Thermo Scientific GeneJET
Qiagen Spin Columns
Silica membrane plus chaotropic salts
No organic extraction
No alcohol precipitation
DNA or RNA, Both
Wide range of clinical samples
Mini, midi, and maxi columns
Vacuum manifold for higher specimen numbers and faster processing
Selected Magnetic Particle Kits
Cortex Biochem MegaZorb
Dynal Dynabeads
DNA DIRECT
Roche Molecular DNA Isolation Kit
BioMerieux NucliSens MINI Mag
Semi-automated; 1-12 specimens (10 l-1 ml)
Boom extraction with magnetic silica particles
Total NA from different sample types (10-50 l)
12 extractions in 35 to 40 min (1 instrument)
24 extractions in 45 to 50 min (2 instruments)
Need for Automated Systems
Significant portion of total testing time and effort is dedicated to sample preparation
Simplification of specimen processing
Need for uniformity and consistency
Direct cost savings
Staffing problems
Automated Sample Processing
Many options are now available Different levels of automation (semi to fully
automated)
Large and small platforms; different sizes, shapes, weights
Varied batch sizes depending on system
Sample processing capacity (1-96 per run)
Various sample input volumes
Variety of chemistries for purification of DNA, RNA or total nucleic acids
Some have post elution protocols/functions
Automated NA Isolation Systems
bioMerieux: mini Mag & easy Mag Extractors
Roche Molecular: MagNA Pure LC, Compact, & 96; AmpliPrep
Qiagen: QIAcube, EZ1 Advanced, EZ1 Advanced XL, QIAsymphony SP
Abbott Molecular: m2000sp Sample Prep Station
Beckman Coulter:
SPRI-TE
NorDiag: Arrow
Promega: Maxwell-16
Invitrogen: iPrep
BioChain: AnaPrep 12
Autogene: QuickGene
610L, 810, 810 Mini
Selected Vendors of Semi and Fully Automated Systems
Compact Systems
Roche MagNA Pure Compact QIAGEN BioRobot EZ1 & EZ1 Advanced/Advanced XL
Beckman Coulter SPRI-TE NorDiag Arrow Promega Maxwell-16
Larger Platforms
BioMerieux NucliSens EASYMAG
Roche MagNA Pure LC 2.0
Roche MagNA Pure 96
Roche AmpliPrep
QIAGEN QIAsymphony SP
Hologic Gen-Probe Panther
Abbott Molecular m2000
Advantages of Automation
Easy/practical to use
Some truly walk away
Reduces hands-on time, sample manipulation
Decreases labor costs, increases throughput
Process variety of sample types and volumes
Standardized technology
Simplified software
Minimize chance of
cross contamination
UV light
decontamination
Minimal maintenance
and down time
Post elution functions
for some
Disadvantages of Automation
Which instrument(s) to choose
Expense
Varied batch sizes depending on system
Varied kit availability for DNA, RNA, total NA
Some samples may require external processing prior to system extraction
Some instruments require manual additions of reagents during extraction
No or slow post elution protocols
Size, shape, and weight
NA Yield, Integrity, Purity
Concentration of DNA 1 A260 U dsDNA = 50 g/ml
1 A260 U ssDNA = 33 g/ml
Purity of DNA Pure DNA: A260/A280 1.8
An A260/A280 < 1.8 (Contaminated with proteins and aromatic reagents)
An A260/A280 > 2.0 RNA contamination
Concentration of RNA
1 A260 U ssRNA = 40 g/ml
Purity of RNA
Pure RNA: A260/A280 2.0
An A260/A280 < 2.0
(Contaminated with
proteins and aromatic
reagents)
Analyzing DNA Analyzing RNA
Storage of Purified Nucleic Acids
Ideally, purified nucleic acids should be used immediately for downstream amplification and detection
If delayed, store under appropriate conditions until testing can be completed
DNA is more stable than RNA and can be stored for long periods at even 4oC in a suitable TRIS buffer
Being more labile, RNA should be stored at -70oC or colder in the same buffer as DNA
As a general rule in my laboratory, storage of all purified nucleic acid preparations are done at
-70oC or colder.
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