NGRL (Wessex) DNA Extraction Evaluation: Chemistries. NC Owen, CJ Mattocks, JF Harvey, NCP Cross
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1 NGRL (Wessex) DNA Extraction Evaluation: Chemistries NC Owen, CJ Mattocks, JF Harvey, NCP Cross
2 Background Autogen NA 3000: Automated nucleic acid isolation system Dec 2002 Full evaluation completed and presented at BSHG York 2003 Report yet to be published Subsequent trials: Chemagen Chemagic Module I ABI 6100 Nucleic Acid PrepStation Qiagen EZ1 BioRobot DRI ChargeSwitch Technology Machery-Nagel Nucleospin 96 Promega Magnesil Integrated systems combining robotic platform and chemistry Individual chemistries set up on a liquid handling robotic platform: Tecan EVO 150
3 Outline Tecan EVO Freedom 150 How we evaluated the gdnas DNA quality DNA quantification PCRs Results / comments DRI chemistry Macherey Nagel chemistry Promega chemistry General conclusions on robot-based chemistries Further testing
4 Tecan Freedom EVO 150 LiHa (Liquid Handling arm) Fixed pipettes (also location for DITI heads) Buffer reservoirs System tubing Hotel (extra plate storage) ROMA (RObotic Manipulating Arm) Tecan GENios Plus (DNA quantifier) Shaker (used in Promega and DRI chemistries) Vacuum Manifold (as used in Machery Nagel Nucleospin 96 chemistry)
5 Tecan cont Liquid handling robotic platform used to assess all robotic based chemistries Flexible platform suitable for many different applications Potential to evolve platform to reflect changing needs User friendly software Accessories and add-ons Vacuum Manifold MN NS96 Shaker Promega, DRI GENios Plus
6 Genomic DNA Quality Theoretical 120ng gdna loaded on 0.6% agarose gel (assuming an expected extraction efficiency of ~30ug DNA per ml of blood) Each sample run against: Lambda (λ) / Hind III ladder 50, 100 and 200ng λ DNA Size / bps 23,130 9,415 6,557 4,361 2,322 2,027 λ / Hind III ladder 50ng 100ng 200ng
7 DNA Quantification Two methods used: PicoGreen assay (fluorescence based) Absorbance Compatible plates for DNA quantification: Fluorescence (requires black, opaque flat bottomed (FB) plates e.g. Greiner, Corning) Luminescence (requires white, FB plates) Absorbance (requires optically clear, UV transparent, FB plates e.g. Greiner, Corning)
8 Tecan GENios Plus Excitation filter slide (contains 4 different filters for 4 wavelength readings) Sample plate Emission filter slide (contains 4 different filters for 4 wavelength readings used for fluorescence and luminescence quantification) Fluorescence, Absorbance, Luminescence Handles assays in microplates, PCR tubes and cuvettes Multilabel reader Top or bottom reading capability
9 PicoGreen Assay Theoretical 50ng gdna (assuming expected extraction efficiency of 30µg DNA per ml of blood) made up to 100µl with 1x TE 100µl PicoGreen (diluted 1/200 with 1x TE) Standard curve created using a range of λ control DNA from 0-1µg/ml RFU gain 70 control y = 36.28x R 2 = conc [ng/ml] Highly correlated line of best fit (R 2 =0.9999) Relationship: Conc. = corrected RFU Blank value = 49 RFU Data used to determine: DNA conc. of sample (ng/µl = µg/ml) Extraction efficiency (µg DNA per ml of blood)
10 Absorbance Optically clear, UV transparent FB plates Tecan GENios Plus Theoretical 2µg DNA (except DRI 200ng) made up to 200µl Readings 260nm, 280nm and 320nm: 260 blank value: blank value: blank value: Light path = 6mm OD of 260nm = 83.33ng/µl 260/280 purity ratio (1.8 ± 0.1) 320 (particulate contamination)
11 Downstream tests: PCR Theoretical 30ng DNA added to each PCR Duplex PCR: AMXY F/R: amelogenin (Xp and Yp11.1) SRY F/R: SRY gene 976bp 787bp 358bp Tests range of amplification up to 1kb
12 Tecan based chemistries Chemistry Machery Nagel NucleoSpin 96 Promega MagneSil DRI ChargeSwitch Tecnology (CST) Extraction method SPE Vacuum based, Silica Membrane MagneSil Paramagnetic Particle technology ph based magnetic beads Blood volume 200µl 5ml 20µl Elution volume 200µl ~1000µl* 100µl Number of Samples per run 96** (2x 36 blood, 2x 12 blank) 8 (6 blood, 2 blank) 48 (36 blood, 12 blank) Total number of Runs 3 (288 samples total) 24 (192 samples total) 9 (432 samples total) Total Blanks tested Total Bloods tested *Exact elution volume calculated by software based on actual volume of blood. All elutions were approximately 1ml **Each run consists of two duplicate extractions of 48 blood samples. Run 1 contained no blanks, whilst runs 2 and 3 contained blanks
13 DRI ChargeSwitch Technology (CST ) Overview Automated partnership with Tecan UK CST magnetic beads Switchable surface charge based on ph of surrounding buffer ph +ve charge binds DNA but not protein/contaminants Impurites washed away in aqueous wash buffer ph (8.5) -ve charge elution of DNA into low salt elution buffer No enzyme inhibiting reagents (e.g. Alcohol, chaotropic salts, organic solvents) Range of extraction protocols 10-20µl blood (manual and automated) - 100µl elution volume µl blood (manual and automated) Buccal cells (manual only) 84 blanks, 348 bloods
14 DRI ChargeSwitch Technology (CST ) Sample Genomic DNAs Trial Run Run 3 = Fail = Blank TOTALS: BLANKS = 86 FAILS = 18/348 (5.2%)
15 DRI ChargeSwitch Technology (CST ) PicoGreen/Absorbance Data PicoGreen Absorbance Elution Conc. (ng/µl) Extraction Efficiency (µg DNA /ml blood) 260/ Elution Conc. (ng/µl) Extraction Efficiency (µg DNA /ml blood) n Min Max Average St Dev FAIL = <5ug/ml extraction rate No. Fails % Fails % 0 0%
16 DRI ChargeSwitch Technology (CST ) Sample Sexing PCRs = Fail = Blank TOTALS: BLANKS = 84 FAILS = 195/348 (56.0%)
17 DRI ChargeSwitch Technology (CST ) Summary Comments Very low volume of blood (20µl) probably too low to ever be used diagnostically ( µl protocol available) CST bead residue (inert?) Unacceptable failure rate (9.8%) Average extraction efficiency ~ ug/ml blood (PG v Abs?) 56.0% PCR failure rate more reflective of problems with initially setting up the protocol on the Tecan Evo 150, not necessarily protocol itself Fine tuning required to reduce alcohol/wash buffer contamination ( downsteam failure? )
18 Macherey Nagel NucleoSpin 96 Blood Overview Silica membrane technology: Blood cells lysed gdna selectively bound to membrane whilst impurities are washed away via vacuum pull through Elution into 96-well plate for storage Vacuum based 200ul blood 100ul elution volume 48 blanks 240 bloods
19 Macherey Nagel NucleoSpin 96 Blood Sample Genomic DNAs 2A Duplicate runs 2B TOTALS: BLANKS = 48 FAILS = 2/240 (0.8%) = Blank = Fail
20 Macherey Nagel NucleoSpin 96 Blood PicoGreen / Absorbance Data PicoGreen Absorbance Elution Conc. (ng/µl) Extraction Efficiency (µg DNA /ml blood) 260/ Elution Conc. (ng/µl) Extraction Efficiency (µg DNA /ml blood) n Min Max Average St Dev FAIL = <5ug/ml extraction rate No. Fails % Fails 6 2.5% 0 0%
21 Macherey Nagel NucleoSpin 96 Blood Sample Sexing PCRs Duplicate runs Duplicate runs TOTALS: BLANKS = 48 FAILS = 2/240 (0.8%)
22 Macherey Nagel NucleoSpin 96 Blood Summary Comments Vacuum manifold very noisy! Vacuum manifold required manual pressure to ensure an acceptable suction (manual removal of blocked wells also required to prevent overflow contamination) Liquid sensing step after each suction to ensure total pull through 2 failed samples (0.8%) due to blocked wells (no DNA expected) average extraction efficiency ~ 20ug/ml blood Good quality gdna (high PCR success rate indicates no contamination no wash/removal step as all wash buffer is pulled though) Vacuum steps need to function in a fully automated way
23 Promega MagneSil TM Blood Genomic Overview MagneSil paramagnetic particles Lysis buffer added Magnetic beads added Lysis buffer / alcohol wash Elution Additional equipment: Shaker Elution incubator Promega tube and magnet rack (4x2 holding 50ml falcon tubes) 5ml blood ~1ml elution volume 48 blanks 144 bloods
24 Promega MagneSil TM Blood Genomic Sample Genomic DNAs T = Blank TOTALS: BLANKS = 48 NO FAILS (0%)
25 Promega MagneSil TM Blood Genomic PicoGreen / Absorbance Data PicoGreen Absorbance Elution Conc. (ng/µl) Extraction Efficiency (µg DNA /ml blood) 260/ Elution Conc. (ng/µl) Extraction Efficency (µg DNA /ml blood) n Min Max Average St Dev FAIL = <5ug/ml extraction rate No. Fails % Fails 5 3.5% 5 3.5%
26 Promega MagneSil TM Blood Genomic Sample Sexing PCRs TOTALS: BLANKS = 48 FAILS = 96/144 (66.7%)
27 Promega MagneSil TM Blood Genomic Summary / Comments 0-3.5% failure rates for gdna gels and both quantification assays suggests generally good DNA extraction, but 66.7% PCR failure rate almost certainly due to buffer/alcohol contamination due to frequent failure to remove all the waste between washes (LiHa alignment in 50ml tubes when removing waste) Average extraction rate ~ µg/ml blood Further evaluation of protocol required (in addition to Macherey Nagel and DRI) with more time to fine tune the robot-chemistry integration
28 PicoGreen Results (Bloods only) Promega MagneSil Machery Nagel NucleoSpin 96 DRI CST Elution Conc. / ng/µl Extraction rate / µg/ml blood Elution Conc. / ng/µl Extraction rate / µg/ml blood Elution Conc. / ng/µl Extraction rate / µg/ml blood n Min Max Average SD No. Fails (<5µg/ml blood) % Fails 0.7% 2.5% 9.8%
29 Summary of Fails Promega MagneSil Machery Nagel NucleoSpin 96 DRI CST gdnas (no visable band) 0% 0.8% 5.2% PicoGreen (<5µg DNA /ml) Absorbance (<5µg DNA /ml) PCRs (no visable band/s) 3.5% 2.5% 9.8% 3.5% 0% 0% 66.7% 0.8% 56.0%
30 General robot-based based chemistry conclusions Setup time Chemistries not ready to go Adjustments / development Months rather than days / weeks Unsuitable diagnostic setup System tubing (Promega or any large volume chemistry) Open blood tubes Lab specific requirements DNA quantification Not straight forward Varying results between techniques Further evaluation required
31 Given time we are confident that all three chemistries could be successfully, reliably and efficiently integrated into a diagnostic laboratory environment
32 Future Testing Further analysis of Absorbance v PicoGreen data Downstream: Further PCRs Multiplex Ligation Probe Amplification (MLPA) Southern blotting
33 Acknowledgements Graeme Daniels Daniel Leach Joanna Johnson Chris Bridge Willem K C van Loon
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