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DNA ExtractionCCDB Protocols for High-Throughput Barcoding
Event Tech Support Number: +1 (610) 431-2465 I Email: [email protected]
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Our Presenter
Natalia Ivanova received her Ph.D. in Molecular Biology from Lomonosov Moscow State University in 1998. Most of her Ph.D. data on the molecular systematics of lichens was gathered at the NMNH, Smithsonian Institution. In 2004 Natalia joined the Hebert laboratory and contributed to the development of cost-effective high throughput barcoding protocols and the integration of robotics into the analytical chain. She currently oversees automation, core lab troubleshooting, the R&D lab, and forensic barcoding at the Canadian Center for DNA Barcoding.
Natalia IvanovaLead DNA Scientist, CCDB
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Our Agenda
• Sampling standardization for high-throughput
• Classification of DNA extraction protocols
• CCDB DNA extraction protocols
• Mix & match Glass Fiber DNA extraction protocol
• Voucher recovery
• Manual vs. automated extraction
• Shipment of DNA and PCR products
• Questions and answers
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direct sampling into microplates
Require subsampling Lab-ready!
2004 2008
tube racks 12x8
High Throughput Sampling Protocols: CCDB Example
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High Throughput Sampling Protocols: Plants
1. Silica gel preserved material2. When sampling into a specimen
array, tissue amount is important 3. A-B – correct4. C-D – incorrect
=
+ 3 4
2
1
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High Throughput Sampling Protocols: FTA cards
FTA FTA Elute
Pathogen inactivation Yes Yes
Room temperature storage and shipment Yes Yes
DNA elution No (only by pH change) Yes
Contaminants Washed away Remain on a filter
Extraction protocol 30 min – 1.5 hours 5 min
Additional reagents Yes No (only water)
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Overview of DNA ExtractionTissue +
Lysis Buffer + ProK
DNA and contaminants released into
solutionAdjust binding
conditionsBind DNA to membrane
1st WashDry the
membrane and elute DNA
Add phenol/chloroform, centrifuge
Transfer top phase, adjust precipitation conditions
Centrifuge to pellet DNA at the bottom
Remove supernatant
Wash DNA with 70%
EtOH
Remove supernatant
Dissolve DNA
56 C
2nd Wash
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Extraction Protocols Not Used At the CCDB
SPRI – magnetic beads
Image from Seradyn web-site
OpenWetWare imageDNA
Contaminants
Size exclusion
Phenol/Chloroform– ethanol precipitation
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Extraction Protocols Used By the CCDB
Bind-wash-elute protocols (membrane based)
Crude lysis (Chelex, alkaline lysis) FTA cards
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CCDB Protocols:Chelex and Alkaline Lysis
• Simple, quick, inexpensive
• Suitable for very small organisms
• Do not produce stable DNA extracts
• Tissue amount is critical
• HotSHOT protocol for rotifers, tardigrades and small crustaceans
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• Lysis – 25-50 µL of 25 mM NaOH, 0.2 mM EDTA, pH 8.0• Incubate for 30 min at 95 C• Neutralization – equal volume of 40 mM Tris-HCl , pH 5.0• Mix, use 1-2 µL in PCR• Store DNA extract for up to 3 years at -20 C
CCDB Protocols:HotShot Alkaline Lysis
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1. Apply sample; dry
2. Punch a disc and place in a tube
3. Rinse punch in water for 5 sec
4. Remove water
5. Use disk in PCR OR add water, heat at 95 C for 15 min; pulse vortex; centrifuge
6. Use eluted DNA in PCR
FTA Elute 96 Well Card – Whatman WB12 9231; Fisher FSSP9777052
CCDB Protocols: FTA Elute Cards
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CCDB Protocols:Glass Fiber DNA Extraction
EcoRI digestion of plant DNA
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Lysis Buffers
• Invertebrate
• Vertebrate
• CTAB
• Algal Buffer (Saunders lab)
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Binding Conditions
Plant Binding Buffer (5M GuSCN buffer)
Binding Mix (3M GuSCN buffer, 50% ethanol)
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Binding Media
1 µm Glass fiber – PALL #5051
3 µm Glass fiber over 0.2 µm Bioinert – PALL #5053
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Invertebrate Extraction • Tissue source – legs or whole specimens
• Invertebrate lysis buffer + Proteinase K
• Incubation overnight at 56 C
• Binding with BM buffer, wash with PWB and WB buffers
• 3 µm GF over 0.2 µm Bioinert membrane
• Elution volume – 30-40 µl
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Vertebrate Extraction • Tissue source – ideally muscle
• Vertebrate lysis buffer + Proteinase K
• Incubation overnight at 56 C
• Binding with BM buffer, wash with PWB and WB buffers
• 1 µm GF membrane
• Elution volume – 50-60 µl
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Plant and Fungal Extraction • Tissue source – silica dried tissue, herbarium
samples, seeds
• Grinding – TissueLyser
• CTAB lysis for 1.5 hour at 65 C
• Binding with 5M GuSCN buffer, wash with BM and WB buffers
• 1 µm GF membrane
• Elution volume – 50-60 µl
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Echinoderm and Mollusk Extraction
• Tissue source – ethanol fixed soft tissue
• CTAB + Proteinase K lysis
• Incubation overnight at 56 C
• Binding with 5M GuSCN buffer, wash with BM and WB buffers
• 3 µm GF over 0.2 µm Bioinert membrane
• Elution volume – 50-60 µl
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Algal DNA Extraction
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Algal Extraction – Saunders Lab/CCDB • Tissue source – silica dried tissue, herbarium• Acetone extraction to remove polyphenols• Grinding – TissueLyser
• Algal buffer + Tween 20 + Proteinase K, incubate 1 hour at room temperature
• Incubate on ice, centrifuge• Transfer aliquot of lysate
• Binding with 5M GuSCN buffer, wash with BM and WB buffers
• 1 µm GF membrane• Elution volume – 50-60 µl
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Voucher Recovery Protocol
Collembola after 12H incubation
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Voucher Recovery Protocol
1. Incubate overnight in a lysis buffer
2. Apply lysate to 350 µl PALL plate with 0.45 µm GHP membrane sitting on top of collection plate with PALL collar
3. Centrifuge at 3000 g for 2 min
4. Use clarified lysate for extraction
5. Add 95% EtOH to lysis and PALL plates
6. Collect vouchers under microscope
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Manual Extraction
• Scalable for 4-6 plates/day
• Centrifugation at 5000 g
• Better for old material
• Less chances of contamination
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Automated & Semi-Automated Extraction
• 20-40 plates/day
• Vacuum manifold
• Centrifugation to remove WB
• Elution in centrifuge
• Possible integration of robotic centrifuge
• Less errors
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PCR Optimization
• Pre-made frozen plates with trehalose• Low dNTPs and primers concentration• Platinum or KAPA Ab Taq polymerase• No PCR cleanup!
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DNA and PCR Products Shipment
• DNA & PCR products exchange between iBOL nodes• Backup system for freezers
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Trehalose and Preservation
1994. Trends Ecol. Evol. 9: 230
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Biomatrica – Emerging Technology
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CCDB: 1 Year Storage Experiment
Mean Contiguous Read Length (bp)
480
500
520
540
560
580
600
Fridge +4 Freezer -20 Biomatrica RT Biomatrica 56 Trehalose RT Trehalose 56
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DNA and PCR Products Recommendations
• Convenient shipment of DNA and PCR products at room temperature
• Backup options for -20 or -80 C archiving
• Trehalose is sufficient for shipment
• Biomatrica is a better choice for forensic, degraded or diluted samples
• More data is needed to advocate transition to dry storage at room temperature
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CCDB Protocols
http://www.dnabarcoding.ca/pa/ge/research/protocols
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Bibliography• deWaard JR, Ivanova NV, Hajibabaei M, Hebert PDN (2008) Assembling DNA barcodes: analytical protocols. In:
Environmental Genomics, Methods in Molecular Biology, vol. 410 (ed. Martin CC), pp. 275-283. Humana Press.• Hajibabaei M, DeWaard JR, Ivanova NV, Ratnasingham S, Dooh RT, Kirk SL, Mackie PM, Hebert PDN (2005) Critical
factors for assembling a high volume of DNA barcodes. Philosophical Transactions of the Royal Society of London B Biological Sciences 360, 1959-1967.
• Ivanova N, Kuzmina M and Fazekas A (2011) CCDB Protocols, Glass Fiber plate DNA extraction for plants, fungi, echinoderms and mollusks. Retrieved from http://www.ccdb.ca/CCDB_DOCS/CCDB_DNA_Extraction-Plants.pdf on April 8, 2011.
• Ivanova NV, DeWaard JR, Hebert PD (2007) CCDB Protocols, Glass Fiber Plate DNA Extraction. Retrieved from http://www.dnabarcoding.ca/CCDB_DOCS/CCDB_DNA_Extraction.pdf on April 7, 2011.
• Ivanova NV, deWaard JR, Hebert PDN (2006) An inexpensive, automation-friendly protocol for recovering high-quality DNA. Molecular Ecology Notes 6, 998-1002.
• Ivanova NV, Fazekas AJ, Hebert PDN (2008) Semi-automated, Membrane-Based Protocol for DNA Isolation from Plants. Plant Molecular Biology Reporter 26, 186-198.
• Ivanova NV, Grainger CM (2007) CCDB Protocols, COI amplification. Retrieved from http://www.dnabarcoding.ca/CCDB_DOCS/CCDB_Amplification.pdf on April 7, 2011.
• Kuzmina M, Ivanova N (2011) CCDB Protocols, PCR amplification for plants and fungi. Retrieved from http://www.dnabarcoding.ca/CCDB_DOCS/CCDB_Amplification-Plants.pdf on April 8, 2011.
• Porco D, Rougerie R, Deharveng L, Hebert PDN (2010) Coupling non-destructive DNA extraction and voucher retrieval for small soft-bodied Arthropods in a high-throughput context: the example of Collembola. Molecular Ecology Resources 10, 942–945.
• Saunders GW (1993) Gel purification of red algal genomic DNA: an inexpensive and rapid method for the isolation of polymerase chain reaction-friendly DNA. Journal of Phycology 29, 251-254.
• Saunders GW (2005) Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications. Philosophical Transactions of the Royal Society B-Biological Sciences 360, 1879-1888.
• Whitlock R, Hipperson H, Mannarelli M, Burke T (2008) A high-throughput protocol for extracting high-purity genomic DNA from plants and animals. Molecular Ecology Resources 8, 736-741.
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Glass Fiber Automated DNA Extraction Video
Launch 7.5 minute video: BiomekFX in Action
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Continuing Supporthttp://www.Barcodeoflife.orghttp://connect.barcodeoflife.orghttp://ibol.org/http://www.boldsystems.org/http://www.dnabarcoding.ca
Recorded presentation will be available at http://connect.barcodeoflife.net/video