FULLY AUTOMATED AND VALIDATED HIGH VOLUME DNA EXTRACTION USING CHEMAGEN MAGNETIC BEADS BASED KITS

FULLY AUTOMATED AND VALIDATED HIGH VOLUME DNA EXTRACTION USING CHEMAGEN MAGNETIC BEADS BASED KITS Tom Janssens & Ivo Salden UZ Gasthuisberg, Center for Human Genetics, Leuven Perkin Elmer Webinar 27th May 2008

OVERVIEW Introduction Validation set-up Results

ISO 15189 accredited since September 2005 History DNA-extraction in Leuven - 2004: Manual salting out extraction 2004-2008: 2008: semi-automated DNA-extraction using a Chemagen-Multiprobe I set-up (manual blood transfer, lysis buffer, protease) End 2007: Validation of automated DNA- extraction using a Chemagen-Multiprobe II Ex with gripper in the context of EUROGENTEST

EuroGentest project Network of excellence funded by the European Commission to improve and harmonize the overall quality in European genetic testing facilities Create validation reports of new technologies Help other labs to implement and validate these technologies

Chemagen principle Introduction Based on the use of paramagnetic beads After lysis of WBC, DNA can bind to the coating of these beads To wash off impurities the beads (together with DNA) can be transferred from one washing buffer to another by inducing a magnetic field The DNA is released from the beads in a final elution step Kits available from 1 µl l to 10 ml blood

Chemagen-Multiprobe I Set-up Manual addition of blood, lysis buffer and protease Multiprobe I: adds all other reagents

Chemagen-Multiprobe II Rewritten the available program to match our old program Customized deck layout enough positions for 3 extraction runs DNA-measurement done off deck barcodes available two 6-way 6 valves Set-up Alternative deck layout (out of this scope) Setup for 2 extraction runs DNA-measurement AND normalization on deck (using Victor³ plate reader)

Validation set-up Starting conditions: 182 anonymized blood samples 5ml of blood per sample Anticoagulant: EDTA Blood samples (< two weeks old) stored at 4 C 4 C or room temperature Using Chemagic DNA Blood Kit special: for separation of DNA from 7 ml whole blood Validation parameters: Well-to to-well contamination Average DNA yield/concentration DNA Purity: OD 260 /OD 280 ratio and OD 260 /OD 230 ratio DNA quality (multiplex drop-out out PCR) DNA degradation Repeatability / reproducibility Testing a range of large blood volumes (1-10ml) 10ml) Extreme storage conditions of blood samples DNA stability DNA performance

Problem during validation process Ethanol problem (1) Problems with some sent out DNA samples Ethanol concentration was over 10%! Comparison with Chemagen samples in other labs 140 120 100 80 60 40 Ethanol concentration (g/l) 20 0 Leuven Nijmegen Brussels Salisbury Older samples Conclusion: : the problem is consistent!

Problem during validation process Ethanol problem (2) Action: the diagnostic validation was stopped Optimization of the extraction protocol in a collaboration between Leuven and Chemagen 140 120 100 80 60 40 20 Adapted protocol from company acetone wash + drying 0 Leuven Nijmegen Brussels Salisbury New protocol [EtOH] (g/l) ~ 1% EtOH

Results

Well-to to-well contamination 4 runs were performed (blood samples alternated with blank (1X PBS) samples) PCR was performed on all samples 1 5 9 M 1 2 3 4 5 6 7 8 9 10 11 12 2 6 10 3 7 11 4 8 12

Yield 182 blood samples were extracted 5 ml of blood WBC was measured for all samples DNA concentration was measured (using Nanodrop and Victor³ plate reader) Yield (µg) Victor³ Yield (µg) Nanodrop 350,0 350,0 300,0 300,0 Yield (µg) Victor³ 250,0 200,0 150,0 100,0 Yield (µg) nanodrop 250,0 200,0 150,0 100,0 50,0 Yield (µg) Victor³ 50,0 Yield (µg) nanodrop 0,0 0,0 5,0 10,0 15,0 20,0 WBC (10E6 cels/ml blood) 0,0 0,0 5,0 10,0 15,0 20,0 WBC (10E6 cels/ml blood) Average yield: 175µg (SD: 46µg) 169µg (SD: 46µg)

Yield

DNA Purity OD 260 /OD 280 ratio OD 260 /OD 230 ratio Average: 1.78 (SD=0.07) Average: 1.69 (SD=0.19)

DNA quality Multiplex drop out PCR (Van Dongen et al. 2003) on all 182 samples M 600 bp 400 bp 300 bp 200 bp 100 bp

DNA degradation 30 random DNA samples analyzed on a 0.8% agarose gel S 1 2 3 4 S: Sizer 1: Stored 16 days before extraction 2: Stored 2 days before extraction 3: Stored 6 days before extraction 4: Stored 0 days before extraction Cell apoptosis/dna degradation is visible when samples are stored for > 4 days

Repeatability same blood sample extracted 3 times in the same run performed on 4 blood samples

Reproducibility same blood sample extracted in 3 different runs on 3 different days by 3 different operators performed on 4 blood samples

DNA stability 8 random DNA samples stored under different conditions for > 1 year: At high temperature 37 C At low temperature 4 C freeze-thaw cycles -20 C C < > < > room temperature 3-Weekly Weekly tested for DNA degradation & Multiplex drop-out out PCR

DNA stability Results immediately after extraction A B C A B C A B C A B C 1 2 3 4 A B C A B C A B C A B C 1 2 3 4 A: Freeze-Thaw cycles B: 4 C C: 37 C Results after 1 year A B C A B C A B C A B C 1 2 3 4 A: Freeze-Thaw cycles B: 4 C C: 37 C A B C A B C A B C A B C 1 2 3 4

Different blood volumes 1 10 ml of blood was extracted during the same run 3 samples were tested 7K Chemagic DNA blood kit was used

Extreme blood storage conditions Old blood samples (6 months at 4 C) 4 Yield: 136,4 µg (6 samples) Ratio 260/280: 1.72 Frozen blood samples Yield: 131,2 µg (2 samples) Ratio 260/280: 1.77 Old samples Frozen sample Fresh samples Old samples Frozen sample

Performance assessment All samples were anonymized and tested for several random diagnostic tests PCR Melting curves MLPA Southern blotting DHPLC Fragment analysis Sequencing Array CGH

Performance assessment Problem with SNP analysis on Taqman Rox signal decreased during run Caused by Cerium-ions ions (carry over from beads production process) Solved by using a Tris elution buffer instead of Tris/EDTA (EDTA is added afterwards) Beads production process has been improved

Other applications used in the lab 200µl l blood extractions (96-well format) (Frozen) placenta extractions (96-well format) (manual lysis - overnight)

PROs Good yield and purity Homogenous DNA after extraction ready to use Fully automatic system including DNA normalization Easy to use Possibility to extract old or frozen blood samples Reliable and robust chemistry Flexibility between small and large blood volume extractions Flexibility in making lab-specific adjustments to the extraction protocol Lysis time Buffer volumes Washing times Possibility to use a bar-coding system

CONs No out-of of-the-box system integration and calibration required Traces of beads present in final DNA sample Large instrument Flammable reagents Gripper errors during validation process Might be solved with more robust Janus system Medium throughput (48 large volume samples/day) Setting up a run requires quite some manual preparations 7 falcon tubes per blood sample Possible traces of cerium ions left in the final DNA sample

Acknowledgements Perkin Elmer Chemagen