FULLY AUTOMATED AND VALIDATED HIGH VOLUME DNA EXTRACTION USING CHEMAGEN MAGNETIC

BEADS BASED KITS

FULLY FULLY AUTOMATEDAUTOMATED AND AND VALIDATEDVALIDATED HIGH VOLUME HIGH VOLUME DNA EXTRACTION USING DNA EXTRACTION USING CHEMAGEN MAGNETIC CHEMAGEN MAGNETIC

BEADS BASED KITSBEADS BASED KITSTom Janssens & Ivo Salden

UZ Gasthuisberg, Center for Human Genetics, Leuven

Tom Janssens & Ivo SaldenTom Janssens & Ivo SaldenUZ Gasthuisberg, Center for Human Genetics, UZ Gasthuisberg, Center for Human Genetics,

Leuven Leuven

Perkin Elmer Webinar27th May 2008

Introduction Introduction

Validation setValidation set--upup

ResultsResults

OVERVIEW

DNADNA--extraction in Leuvenextraction in Leuven…… -- 2004: Manual salting out extraction2004: Manual salting out extraction

20042004--2008: semi2008: semi--automated DNAautomated DNA--extraction extraction using a Chemagenusing a Chemagen--Multiprobe I setMultiprobe I set--upup

(manual blood transfer, lysis buffer, protease)(manual blood transfer, lysis buffer, protease)

End 2007: Validation of automated DNAEnd 2007: Validation of automated DNA--extraction using a Chemagenextraction using a Chemagen--Multiprobe II Ex Multiprobe II Ex with gripper in the context of EUROGENTESTwith gripper in the context of EUROGENTEST

ISO 15189 accredited since September 2005ISO 15189 accredited since September 2005

HistoryHistory

EuroGentest projectEuroGentest projectNetwork of excellenceNetwork of excellence funded by the European funded by the European Commission to improve and harmonize the overall quality Commission to improve and harmonize the overall quality in European genetic testing facilitiesin European genetic testing facilitiesCreate Create validation reports validation reports of new technologiesof new technologiesHelp other labs to Help other labs to implement and validateimplement and validate these these technologiestechnologies

IntroductionIntroductionChemagen principleChemagen principle

Based on the use of Based on the use of paramagnetic beadsparamagnetic beadsAfter After lysislysis of WBC, DNA can of WBC, DNA can bind to the coatingbind to the coating of these beadsof these beadsTo To wash off impurities wash off impurities the beads (together with DNA) can the beads (together with DNA) can be transferred from one washing buffer to another by inducing a be transferred from one washing buffer to another by inducing a magnetic fieldmagnetic fieldThe DNA is released from the beads in a final The DNA is released from the beads in a final elution elution stepstepKits available from 1 Kits available from 1 µµl to 10 ml bloodl to 10 ml blood

Manual addition of blood, lysis buffer and Manual addition of blood, lysis buffer and proteaseproteaseMultiprobe I: adds all other reagentsMultiprobe I: adds all other reagents

ChemagenChemagen--Multiprobe IMultiprobe ISetSet--upup

Rewritten the available program to match our old program Rewritten the available program to match our old program

Customized deck layoutCustomized deck layoutenough positions for 3 extraction runsenough positions for 3 extraction runsDNADNA--measurement done off deckmeasurement done off deckbarcodes availablebarcodes availabletwo 6two 6--way valvesway valves

Alternative deck layout (out of this scope)Alternative deck layout (out of this scope)Setup for 2 extraction runsSetup for 2 extraction runsDNADNA--measurement AND normalization on deck (using Victormeasurement AND normalization on deck (using Victor³³ plate reader)plate reader)

ChemagenChemagen--Multiprobe II Multiprobe II SetSet--upup

Validation setValidation set--upupStarting conditionsStarting conditions::

•• 182 anonymized blood samples182 anonymized blood samples•• 5ml of blood per sample5ml of blood per sample•• Anticoagulant: EDTAAnticoagulant: EDTA•• Blood samples (< two weeks old) stored at 4Blood samples (< two weeks old) stored at 4°°C or room temperatureC or room temperature•• Using Chemagic DNA Blood Kit special: for separation of DNA fromUsing Chemagic DNA Blood Kit special: for separation of DNA from 7 ml 7 ml

whole bloodwhole blood

Validation parametersValidation parameters::•• WellWell--toto--well contaminationwell contamination•• Average DNA yield/concentrationAverage DNA yield/concentration•• DNA Purity: DNA Purity: ODOD260260/OD/OD280 280 ratio and ratio and ODOD260260/OD/OD230 230 ratioratio•• DNA quality (multiplex dropDNA quality (multiplex drop--out PCR)out PCR)•• DNA degradationDNA degradation•• Repeatability / reproducibilityRepeatability / reproducibility•• Testing a range of large blood volumes (1Testing a range of large blood volumes (1--10ml)10ml)•• Extreme storage conditions of blood samplesExtreme storage conditions of blood samples•• DNA stabilityDNA stability•• DNA performanceDNA performance

Problem during validation processProblem during validation processEthanol problem (1)Ethanol problem (1)

Problems with some sent out DNA samplesProblems with some sent out DNA samplesEthanol concentration was over 10%!Ethanol concentration was over 10%!ComparisonComparison with Chemagen samples in other labswith Chemagen samples in other labs

ConclusionConclusion: the problem is consistent!: the problem is consistent!

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Leuven Nijmegen Brussels Salisbury

Ethanolconcentration (g/l)

Older samples

Problem during validation processProblem during validation processEthanol problem (2)Ethanol problem (2)

Action: the diagnostic validation was Action: the diagnostic validation was stoppedstoppedOptimizationOptimization of the extraction protocol in a of the extraction protocol in a collaboration between Leuven and Chemagencollaboration between Leuven and Chemagen

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Leuven Nijmegen Brussels Salisbury Newprotocol

[EtOH](g/l)

Adapted protocol from companyacetone wash + drying

~ 1% EtOH

ResultsResults

WellWell--toto--well contaminationwell contamination4 runs were performed 4 runs were performed (blood samples alternated with blank (1X PBS) samples)(blood samples alternated with blank (1X PBS) samples)

PCR was performed on all samplesPCR was performed on all samples

M 1 2 3 4 5 6 7 8 9 10 11 12M 1 2 3 4 5 6 7 8 9 10 11 121 5 9 1 5 9

2 6 10 2 6 10

3 7 11 3 7 11

4 8 12 4 8 12

YieldYield182182 blood samples were extracted blood samples were extracted 5 ml of blood5 ml of bloodWBC was measured for all samplesWBC was measured for all samplesDNA concentration was measured DNA concentration was measured (using Nanodrop and Victor(using Nanodrop and Victor³³ plate reader)plate reader)

Average yield: Average yield: 175175µµgg (SD: 46(SD: 46µµg) g) 169169µµg g (SD: 46(SD: 46µµg)g)

Yield (µg) Victor³

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50,0

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250,0

300,0

350,0

0,0 5,0 10,0 15,0 20,0

WBC (10E6 cels/ml blood)

Yiel

d (µ

g) V

icto

r³

Yield (µg) Victor³

Yield (µg) Nanodrop

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WBC (10E6 cels/ml blood)

Yiel

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g) n

anod

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Yield (µg) nanodrop

YieldYield

DNA PurityDNA PurityOD260/OD280 ratio OD260/OD230 ratio

Average: Average: 1.78 (SD=0.07) Average: Average: 1.69 (SD=0.19))

DNA qualityDNA quality

Multiplex drop out PCR (Van Dongen et al. 2003) on Multiplex drop out PCR (Van Dongen et al. 2003) on all 182 samplesall 182 samples

M

600 bp

400 bp

300 bp

200 bp

100 bp

DNA degradationDNA degradation30 random DNA samples analyzed on a 0.8% agarose 30 random DNA samples analyzed on a 0.8% agarose gelgel

Cell apoptosis/DNA degradation is visible when samples are stored for > 4 days

S 1 2 3 4

S: Sizer1: Stored 16 days before extraction2: Stored 2 days before extraction3: Stored 6 days before extraction4: Stored 0 days before extraction

RepeatabilityRepeatabilitysame blood sample extracted 3 times in the same runsame blood sample extracted 3 times in the same run

performed on 4 blood samplesperformed on 4 blood samples

ReproducibilityReproducibilitysame blood sample extracted in 3 different runs on 3 same blood sample extracted in 3 different runs on 3 different days by 3 different operatorsdifferent days by 3 different operators

performed on 4 blood samplesperformed on 4 blood samples

8 random DNA samples stored under different conditions for > 1 8 random DNA samples stored under different conditions for > 1 year:year:

At high temperature At high temperature 3737°°CCAt low temperature At low temperature 44°°C C freezefreeze--thaw cyclesthaw cycles --2020°°C <C <––> room temperature> room temperature

33--Weekly tested for DNA degradation & Multiplex dropWeekly tested for DNA degradation & Multiplex drop--out PCRout PCR

DNA stabilityDNA stability

Results immediately after extractionResults immediately after extraction

Results after 1 yearResults after 1 year

1 2 3 4 A B C A B C A B C A B C

A: Freeze-Thaw cycles

B: 4°C

C: 37°C

DNA stabilityDNA stability

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

1 2 3 4 A B C A B C A B C A B C

A B C A B C A B C A B C 1 2 3 4

Different blood volumesDifferent blood volumes

1 1 –– 10 ml of blood was extracted during the same run10 ml of blood was extracted during the same run3 samples were tested3 samples were tested7K Chemagic DNA blood kit was used7K Chemagic DNA blood kit was used

Extreme blood storage conditionsExtreme blood storage conditionsOld blood samples (6 months at 4Old blood samples (6 months at 4°°C)C)

•• Yield: 136,4 Yield: 136,4 µµg g (6 samples)(6 samples)

•• Ratio 260/280: 1.72Ratio 260/280: 1.72

Frozen blood samplesFrozen blood samples•• Yield: 131,2 Yield: 131,2 µµg g (2 samples)(2 samples)

•• Ratio 260/280: 1.77Ratio 260/280: 1.77

Old samples Frozen sampleOld samples Frozen sample Fresh samples

Performance assessmentPerformance assessmentAll samples were anonymized and tested All samples were anonymized and tested for several for several randomrandom diagnostic testsdiagnostic tests

•• PCRPCR•• Melting curvesMelting curves•• MLPAMLPA•• Southern blottingSouthern blotting•• DHPLCDHPLC•• Fragment analysisFragment analysis•• SequencingSequencing•• Array CGHArray CGH•• ……

Performance assessmentPerformance assessmentProblem with SNP analysis on TaqmanProblem with SNP analysis on Taqman

•• Rox signal decreased during runRox signal decreased during run•• Caused by CeriumCaused by Cerium--ions ions

(carry over from beads production process)(carry over from beads production process)

•• Solved by using a Tris elution buffer instead of Solved by using a Tris elution buffer instead of Tris/EDTA Tris/EDTA (EDTA is added afterwards)(EDTA is added afterwards)

•• Beads production process has been improvedBeads production process has been improved

Other applications used in the labOther applications used in the lab

200200µµl blood extractions (96l blood extractions (96--well format)well format)

(Frozen) placenta extractions (96(Frozen) placenta extractions (96--well well format)format) (manual lysis (manual lysis -- overnight) overnight)

PROsPROs•• Good yield and purityGood yield and purity•• Homogenous DNA after extraction Homogenous DNA after extraction ready to useready to use•• Fully automatic system including DNA normalizationFully automatic system including DNA normalization•• Easy to useEasy to use•• Possibility to extract old or frozen blood samples Possibility to extract old or frozen blood samples •• Reliable and robust chemistryReliable and robust chemistry•• Flexibility between small and large blood volume Flexibility between small and large blood volume

extractionsextractions•• Flexibility in making labFlexibility in making lab--specific adjustments to the specific adjustments to the

extraction protocolextraction protocolLysis timeLysis timeBuffer volumesBuffer volumesWashing timesWashing times……

•• Possibility to use a barPossibility to use a bar--coding systemcoding system

CONsCONs•• No outNo out--ofof--thethe--box system box system integration and calibration integration and calibration

requiredrequired•• Traces of beads present in final DNA sampleTraces of beads present in final DNA sample•• Large instrumentLarge instrument•• Flammable reagentsFlammable reagents•• Gripper errors during validation processGripper errors during validation process

Might be solved with more robust Janus systemMight be solved with more robust Janus system

•• Medium throughput (48 large volume samples/day)Medium throughput (48 large volume samples/day)•• Setting up a run requires quite some manual preparationsSetting up a run requires quite some manual preparations

7 falcon tubes per blood sample7 falcon tubes per blood sample

•• Possible traces of cerium ions left in the final DNA samplePossible traces of cerium ions left in the final DNA sample

AcknowledgementsAcknowledgementsPerkin ElmerPerkin ElmerChemagenChemagen