APPROACHES TO NUCLEIC ACID EXTRACTION
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1 APPROACHES TO NUCLEIC ACID EXTRACTION Richard L. Hodinka, Ph.D. University of South Carolina School of Medicine Greenville Greenville Health System, Greenville, SC
2 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
3 Ingredients Needed for Success with Molecular Assays Specimens NA Isolation Reagents Facilities Amplification Detection Quality Control Quality Assurance Result Analysis & Interpretation
4 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
5 Practical Rules of Thumb As a general rule, submit specimens to laboratory as quickly as possible after collection Keep cold (2-8 o C) for short-term transit and storage; avoid extend times at RT Freeze to at least -70 o C 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
6 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
7 RNA Extraction Can Be Frustrating
8 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?
9 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
10 Bead Beating Cell/Tissue Disruption Grinding Beads Shaking Homogenizers Physical disruption before extraction
11 MagNA Lyser Cell/Tissue Disruption
12 Old School-Traditional Methods
13 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
14 Organic Extraction Methods Accomplished using an organic mixture of phenolchloroform Nucleic acid separates into an aqueous phase, lipids in the organic phase, and proteins at the interphase Keeping the ph at and adding isoamyl alcohol would help keep RNA in aqueous phase openwetware.org/wiki/phenol/chloroform_extraction
15 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
16 New Wave Technology
17 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
18 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
19 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
20 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
21 Spin Column Technology A B C D Lysis Bind Wash Elute
22 Selected Commercial Spin Columns Qiagen Clontech Amresco Gentra Systems Stratagene Roche Molecular Thermo Scientific QIAmp, RNeasy NucleoSpin Cyclo-Prep Generation Capture Strata Prep High Pure GeneJET
23 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
24 Qiagen QIAcube Spin-Column Processing
25 QIAcube HT 96 Well Platform
26 Magnetic Particle Technology
27 Selected Magnetic Particle Kits Cortex Biochem Dynal Roche Molecular MegaZorb Dynabeads DNA DIRECT DNA Isolation Kit
28 Magnetic Separators
29 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)
30 Akonni TruTip Technology < 7 min extraction
31 For Those Who Desire A Bit More
32 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
33 The Modern Family of Instruments
34 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
35 Automated NA Isolation Systems Selected Vendors of Semi and Fully Automated 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
36 Compact Models
37 Compact Systems Roche MagNA Pure Compact QIAGEN BioRobot EZ1 & EZ1 Advanced/Advanced XL Beckman Coulter SPRI-TE NorDiag Arrow Promega Maxwell-16
38 Luxury Sedans Bugatti 16C Galibier
39 Larger Platforms Roche MagNA Pure LC 2.0 BioMerieux NucliSens EASYMAG Abbott Molecular m2000 Roche MagNA Pure 96 QIAGEN QIAsymphony SP Hologic Gen-Probe Panther Roche AmpliPrep
40 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
41 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
42 NA Yield, Integrity, Purity Analyzing DNA Concentration of DNA 1 A 260 U dsdna = 50 g/ml 1 A 260 U ssdna = 33 g/ml Purity of DNA Pure DNA: A 260 /A An A 260 /A 280 < 1.8 (Contaminated with proteins and aromatic reagents) An A 260 /A 280 > 2.0 RNA contamination Analyzing RNA Concentration of RNA 1 A 260 U ssrna = 40 g/ml Purity of RNA Pure RNA: A 260 /A An A 260 /A 280 < 2.0 (Contaminated with proteins and aromatic reagents)
43 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 4 o C in a suitable TRIS buffer Being more labile, RNA should be stored at -70 o C 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 -70 o C or colder.
44 It s All in the Extraction
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