Sample Collection System for DNA Analysis of ... - … of Justice and prepared the following final...

The author(s) shown below used Federal funds provided by the U.S. Department of Justice and prepared the following final report: Document Title: Sample Collection System for DNA Analysis of

Forensic Evidence: Towards Practical, Fully‐Integrated STR Analysis

Author: Eugene Tan Document No.: 236826

Date Received: December 2011 Award Number: 2008-DN-BX-K010 This report has not been published by the U.S. Department of Justice. To provide better customer service, NCJRS has made this Federally-funded grant final report available electronically in addition to traditional paper copies.

Opinions or points of view expressed are those

of the author(s) and do not necessarily reflect the official position or policies of the U.S.

Department of Justice.

SampleCollectionSystemforDNAAnalysisofForensicEvidence NetBioInc.NIJAward2008‐DN‐BX‐K010

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SampleCollectionSystemforDNAAnalysisofForensicEvidence:TowardsPractical,Fully‐

IntegratedSTRAnalysisEugeneTan

NetBio,830WinterSt,Waltham,MA02451

This project was supported by Grant Number NIJ 2008-DN-BX-K010 awarded by the National

Institute of Justice, Office of Justice Programs, US Department of Justice.

I. ExecutiveSummary

The goal of this research project was to develop a sample collection system for law

enforcementagentstocollect,protect,anddocumentbiologicalevidenceandtoperforminitial

processing steps ina format compatiblewith rapid sample‐in to results‐outmicrofluidicDNA

analysis. The sample collection system consists of an evidence collection device (a collecting

swab and accompanying tube for swab insertion) and a sample processing cartridge. This

“Smart CartridgeTM” will lyse cells, solubilize and concentrate DNA, and transfer the DNA

solution to a microfluidic biochip. The microfluidic biochip will process the DNA solution to

generatepurifiedDNAcompatiblewiththerequirementsforSTRanalysis.

Theevidencecollectiondevicehasbeendesignedtoacceptblood,buccalcells, saliva,

andcellularsamples.Cotton,modifiedcellulose,foam,nylon,polyesterandrayon‐tippedswabs

wereevaluatedforreproducibilityofcellcollection,easeofcollection,tolerancetocollection

This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.


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protocol, liquidabsorption,andapplication in forensicapplications.TheBodeSecurSwabwas

selectedforthesamplecollectionsystem.

To operate the system, the userwill collect a swab sample, insert the swab into the

accompanying collection tube, place the tube into the Smart Cartridge, and press a start

button—nofurtheruserinterventionisrequired.TheDNAgeneratedbythesamplecollection

systemwill thenbe transferredautomatically toNetBio’s fully integratedmicrofluidicbiochip

forSTR typing.Severalapproaches to thesampleprocessingstepswereevaluatedand those

selectedwereintegratedtodevelopanoptimizedSmartCartridge.

Thisresearchhasresultedinthedevelopmentofacrimesceneevidencecollectionand

processing system compatible with an instrument that will generate an STR profile in 45

minutes from sample introduction with minimal operating requirements. The integrated

instrument will process 16 samples in parallel and dramatically reduce the costs (including

labor, space, andvalidation)of settingupandoperatingaDNA lab. Theevidence collection

device and Smart Cartridge are designed such that the system will be easy to operate and

compatible with both forensic and microfluidic requirements. This sample collection system

representsacriticaladditiontoafullyintegratedsystemforforensicDNAanalysis.



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II. TableofContents

I. ExecutiveSummary ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐1

II. TableofContents ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐3

III. Introduction‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐5

A. Purpose,Goals,andObjectives‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐5

B. NetBioSample‐intoResults‐outComponents ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐8

1. SamplecollectionandDNArecovery ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐8

2. MicrofluidicDNAExtractionandPurification ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 10

3. RapidMultiplexPCRAmplificationinaMicrofluidicChip ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 13

4. MicrofluidicSeparationandDetection ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 16

C. ReviewofRelevantLiterature‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 19

D. RequirementsofaSampleCollectionSystem‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 23

IV. ResearchDesignandMethods‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 24

A. OverviewofResearchDesign ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 24

B. MaterialsandMethods ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 25

1. MockCaseworkandReferenceSamples. ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 25

2. VortexExtractionofDNAfromSwabs. ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 25

3. MechanicalAgitationforExtractionofDNAfromSwabs. ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 26

4. MicrofluidicBiochipPurification. ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 26

5. AutomatedMicrofluidicExtractionandPurificationofDNAfromSwabsinSmart

Cartridge. ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 26

6. STRAmplificationReaction. ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 27



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7. STRseparationanddetectioninstrumentation ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 27

V. DisseminationStrategy ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 27

VI. ImplicationsforCriminalJusticePolicyandPractice ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 28

VII.ResultsandDiscussions ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 29

A. Evidencecollectionmedia ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 29

B. ChemicalLysis ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 32

C. SmartCartridgeProcessStep2:SelectionofSilicaFiberMembrane‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 34

D. SmartCartridgeDevelopment ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 35

E. TandemEvaluationandOptimizationoftheThreeProcessSteps ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 36

F. TestingoftheEvidenceCollectionDeviceandSmartCartridge ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 37

VIII. Conclusions ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 41



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III. Introduction

A. Purpose,Goals,andObjectives

Theuseofmicrofluidicdevices limitsthesamplesforanalysisto liquids,andtheminiaturized

natureof thedevices limits thevolumeof sample that canbeanalyzed.These limitationson

microfluidic sample format preclude the direct insertion of biological samples collected by

validated forensic collection methods directly into a microfluidic biochip. To microfluidically

process a conventionally collected biological sample, extensivemanual processing would be

requiredtoextract,solubilize,andconcentratetheDNAofinterest.

Thepurposeof thisprojectwas todevelopa sample collection system thatwill allow

law enforcement agents to easily collect, protect and document biological evidence, and to

performrapidsample‐intoresults‐outmicrofluidicDNAanalysisintheforensicslaboratoryor

at the crime scene. Accordingly, this research focused on the development of an evidence

collection device and a sample processing cartridge (termed the “Smart Cartridge”) thatwill

operateintandemwithafullyintegratedforensicinstrumentandmicrofluidicbiochipthatwill

perform DNA extraction and purification, STR amplification, andmicrofluidic separation and

detection.

The process flow of the fully integrated STR analysis system is shown in Figure 1. A

forensicsampleiscollectedusingtheevidencecollectiondeviceandthenplacedintotheSmart

Cartridge. After pressing the start button, all required operations are completed, and STR

resultsarereported.Withinthecartridge,severalprocessingstepsareaccomplished(celllysis,

DNA solubilization, and concentration; Figure 2). On the biochip, the DNA is subjected to



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purification(ModuleI),andanappropriatealiquotoftheremainingDNAistransferredtothe

PCR chamber for STR amplification (Module II). The DNA fragments are then subjected to

separationanddetection(ModuleIII),resultinginanSTRprofile.

Figure1.ProcessflowforsampleintoresultsoutSTRanalysisofcaseworksamplesinthefullyintegratedsystem.



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Figure2.ProcessflowontheEvidenceCollectionDeviceandSmartCartridge.

Finally, commercially available STR typing kits allow the effective generationof highly

accurateSTRprofiles,butonlywhentheinputDNAtemplatefallswithinanarrowlyoptimized

range.TheDNAadvisoryboard to theFederalBureauof Investigationhas recommendedthe

use of human‐specific DNA quantitation prior to PCR amplification of casework samples as

thereisthepotentialforsamplecontaminationfromnon‐humansourcesincludingnon‐human

mammalian,bacterial, and fungalDNA.Accordingly,a fully‐integratedmicrofluidic system for

forensic human identification should perform human‐specific DNA quantitation in order to

determinepreciselytheamountofDNAtemplatetobesubjectedtoSTRamplification.NetBio

is pursuing microfluidic quantitation under NIJ Award 2008‐DN‐BX‐K009, “RapidMicrofluidic

HumanSpecificDNAQuantitation.”



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B. NetBioSampleintoResultsoutComponents

1. SamplecollectionandDNArecovery

Bode Technology has developed numerous highly advanced and innovative DNA collection

productsincludingtheCrimeScenecollectorandSliderBuccalDNACollector.TheCrimeScene

collectorwasspecificallydesignedtoprovide lawenforcementsecuremorereliableevidence

fromcrimescenes, toprotect thecollectedsamples forshippingandstorage,andtosimplify

documentation of biological evidence. Evaluation of sample collection methods show that

swabbing is more effective for collection and recovery of DNA compared to taping.

Furthermore,cottontippedswabsarethemosteffectivemediaandformatforcollectionand

recoveryofDNA(Figure3).

NetBio previously performed an initial evaluation of lysis solution formulations.

Increases in the DNA extraction efficiency of 1.2 to 2 times compared to that of the

unoptimizedlysissolutionswereachieved.Figure4showstheDNApurificationefficiencyfor10

– 300µl of humanblood in eachof five lysis solution formulations. In this earlier research,

optimizationofthecollectionmediaandlysisbuffer,intandemoverarangeofsampletypes,

was performed tomaximizeDNA recovery.Ultimately, formulations based on a guanidinium

chloride(GuHCl)protocolwereselectedforfurtheroptimization.



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Figure3.DNArecoveryforepithelialcellsonpolyesterblendfabriccollectedusingvarioussamplecollectionmatricesanddevices.



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Figure4.ComparisonofGuHClandSDSNaClbasedlysissolutionsforextractionofDNAfromwholeblood.

2. MicrofluidicDNAExtractionandPurification

Development of themicrofluidic biochip‐based extraction and purificationmodule has been

completed,andthisworkwasfundedinpartbyNIJAward2007‐DN‐BX‐K184.Preliminarywork

hasbeendemonstratedusing8‐samplebiochips incorporatinga solidphasebind/wash/elute

membrane, and a schematic of the current biochip is shown in Figure 5. For blood, lysis is

performed with a GuHCl‐based‐lysis buffer, and DNA in the lysate is bound to a silica

membrane. The operations to manipulate the fluids through the biochip are driven in an

automated fashion by the sequential actuation of pneumatic lines. Figure 6 shows that the

humangenomicDNApurifiedonthebiochiphasanaveragelengthofapproximately50kbp,a



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size appropriate for STR amplification. Finally, DNA extracted using thismicrofluidic biochip

amplifies effectively and generates STR profiles (Figure 7) that are indistinguishable from

conventionaltubebasedreactions.

Figure5.Schematicofan8samplemicrofluidicchipforextractionandpurificationofDNA.



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Figure6.AgarosegelanalysisofDNAextractedfromwholebloodwithmicrofluidicbiochip.(1:Biochipextaction,2:Qiagencontrolextraction,λ:50kbplambdaDNA)

Figure7.STRprofilefromDNAextractedandpurifiedfromwholebloodwithmicrofluidicbiochip.



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3. RapidMultiplexPCRAmplificationinaMicrofluidicChip

Rapid multiplexed STR amplification was accomplished by focusing on two major areas:

rigorous optimization of all reaction mix components and cycling parameters and the

developmentof instrumentationtoallowrapidandhighly‐controlledtemperaturetransitions.

The custom thermal cycler shown in Figure 8 is designedwith a high output thermoelectric

cooler/heatermounted toahighefficiencyheat sink, together referred toas theheatpump.

This instrumentacceptsa16‐chambermicrofluidicbiochip (Figure9)which is coupled to the

heatpumpbyapplyingacompressivepressurewithaclampingmechanism.EachPCRchamber

holds 7µl of PCR reaction solution. The 16 PCR reaction solutions are placed into individual

chambers of the microfluidic biochip. The thermal cycler has the ability to heat and cool a

reactionsolutionatratesof15.8°C/sand15.4°C/srespectively,muchfasterthancommercially

available cyclers. Appropriate selection of an enzymes allows a highly multiplexed PCR

reactiontobeperformedinaslittleas17minutes(Figure10).Figure11showsarepresentative

fastSTRprofileusingtheNetBiothermalcyclerandseparatedanddetectedonGenebenchFX.

The fast PCR profiles generated using this approachmeet forensically relevant requirements

including signal strength, stutter, peak‐height ratio, complete non‐template nucleotide

addition,andinterlocusbalance(Giese2009).Figure12showstherelationshipbetweenDNA

template level and signal strength, indicating that the amplification systemhas a near single

copylimitofdetection.



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Figure8.NetBiofastthermalcycler.

Figure9.16samplePCRBiochipforrapidhighlymultiplexedamplificationinNetBio’sfastthermalcycler.

1

cm



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Figure10.ThermalprofileforfastmultiplexedamplificationofSTRsinthefastthermalcycler.

Figure11.STRprofilefor0.5ngDNA(9947A)amplifiedinbiochipunderfastthermalcyclingconditionswithprimersfromtheAmpFlSTRProfilerPlusIDPCRamplificationkit.



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Figure12.SignalstrengthofvWA17and18,amplifiedinbiochipunderfastthermalcyclingconditionswiththefastthermalcyclerandseparatedanddetectedonGenebenchFX.

4. MicrofluidicSeparationandDetection

Genebench FXTM is a rapid, high resolution, and high sensitivity DNA fragment sizing and

sequencing instrument for laboratoryandfielduse. The instrumentseparatesDNAbasedon

fragment size by electrophoresis on microfluidic biochips, and excitation and detection of

labeledDNA fragments isaccomplishedby laser‐induced fluorescencedetection. Genebench

FX(Figure13)canbeoperatedinboththeforensiclaboratoryandinthefield,haslowpower

consumption,andisCEmarkedundertheLowVoltageDirective73/23/EEC.Separationofthe

DNAfragmentstakeplacewithinamicrofluidicbiochipthatisfilledwithasievingmatrix.The

biochip(Figure14)accepts16samplestoallowforsimultaneousanalysisofmultiplesamples

andrequiredcontrolreactions.



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DNA samples for analysis by Genebench‐FX are preparedwith conventionalmethods

andmanualpipettingintothesamplereservoirsofthebiochip.Afterloadingthesamplesand

buffers, thebiochip isplaced intothe instrumentandanalysiscontinueswithout furtheruser

manipulation, with electric fields applied to electrophoretically separate the DNA and

excitementanddetectionof the fluorophoresas theypass through thedetection zone. The

fully integrated version of the Genebench instrument, the focus of NetBio’s development

efforts,willfeatureafullyintegratedmicrofluidicbiochipthatwillacceptforensicsamplesfrom

the user and perform all DNAmanipulations, allowing for sample‐in to results‐out operation

withouttheneedforuserintervention.

Figure15showsanSTRprofileusingtheSGM+primerset(AppliedBiosystems,Foster

City,CA)generatedonGenebench‐FX.ThePCRproductresultedfromtheamplificationof1ng

of007DNAtemplateunderthemanufacturer’srecommendedcondition.Genebench‐FXhas5

color detection capability, allowing the instrument to perform 5 color multiplexed DNA

fragmentsizingassayssuchasthoserequiredbytheAmpFlSTRIdentifilerPCRAmplificationKit

(AppliedBiosystems).



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Figure13.GenebenchFXseries100.Arrowindicatessiteofmicrofluidicbiochipinsertion.

Figure14.16samplemicrofluidicchipforseparationanddetectiononGenebenchFX.

Cathode Anode

SampleandWasteWells

SeparationChannel

ExcitationandDetectionRegion



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Figure15.GenebenchFXSTRprofileof1ngDNA(007)withAmpFlSTRSGM+amplificationkit.

Takentogether,theseresultsshowthedevelopmentalprogressofthemodulesrequiredforafullyintegrated,sample‐intoresults‐outforensicDNAanalysissystem.

C. ReviewofRelevantLiterature

SampleCollectionandElution. Collectionofbiological evidence forDNAanalysis fromcrime

scenes is a process that effectively collects cells from a variety of surfaces, preserves the

collected cells to preventmolecular degradation, and releases thematerial for downstream

processing. Blood, semen, epithelial cells, urine, saliva, bone, and various tissues can be

associatedwiththecrimesceneandrequirecarefulandeffectivecollection(Lee1998).Theage

of the sample as well as the surface on which it is found are factors determining how the

sampleshouldbecollected.



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Themostcommoncollectiontechniqueisperformedusingacottonswab.Asingleswab

canbetakenfromanareaorthewet‐drydoubleswabtechniquecanbeused.Thedoubleswab

technique may be the most prevalent, and a number of different fluids including water,

buffered saline, or lysis buffers can be used tomoisten the first swab (Leemans 2006). This

technique allows for dried samples to become re‐hydrated, with the majority of material

collectedonthefirstswabandthedrysecondswabcollectingtheremainderofthesample.

Swabs can be comprised of variousmaterials including cotton or synthetic variations.

Swabbing can also be performed using gauze‐like materials, disposable brushes, or

commercially available biological sampling kits (Lauk and Schaaf 2007). Another standard

collection technique involves taking cuttings of the area of interest such as a biological fluid

fromclothing;howeverthisdestroystheintegrityoftheevidence.Adhesivetapeliftsarealso

usedonavarietyofsurfacestocollecttraceevidencethatmaycontainhumanDNA.

Ifsamplesarecollectedandarenotprocessedimmediately,theyshouldbeallowedto

dry to prevent fungal or bacterial growth. Evidentiary samples should not be immediately

sealed in plastic, which can result in microbial growth and cause degradation of the DNA.

Typically,swabsorcuttingsareplaced inbreathablecontainersmadeofpaperorcardboard.

Storingcollectedevidenceincool,dryenvironmentsalsominimizessampledeterioration(Lee

andLadd2001).

Onceasampleiscollected,thenextchallengeistoremovethebiologicalcellsfromthe

collectionmatrices.Cellscanbeextracteddirectlyfromthecollectiondevice,asisthecasein

mostcuttingsorcottonswabs,byplacingthecollectorintoanextractionbuffer.Inthecaseof



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tape lifts, swabs moistened with solutions such as xylene can be utilized to dissolve the

adhesive,collectingasmuchmaterialfromthetapeaspossiblepriortosubjectingtheswabto

extraction.

In some cases,DNA from the collected cells should not be extracteddirectly and the

biological material should be removed first for examination. When using cotton swabs to

collectmaterial,therecanbeproblemsremovingbiologicalmaterialfromthecottonmatrix;as

the cotton swab dries after collection, the biological material can adhere to the swab. For

example, due to the saccharic composition of the spermatocyte membrane, spermatocytes

stick to solid supports, especially cotton (Lazzarino 2008). In order to release themaximum

amountofmaterialfromtheswabs,avarietyofbuffershavebeentestedandcomparedtothe

standarddifferentialextractionbuffer.Useofdetergentssuchas1‐2%sodiumdodecylsulfate

(SDS) has shown to increase sperm cell recovery (Norris 2007). Also, the addition of low

amounts of cellulase has shown to releasemore epithelial and sperm cells from the cotton

swabmatrixthanbufferelutionalone(Voorhees2006).

Problematic Samples. There can be many challenges to obtaining STR profiles from

biological materials including low quantity or quality of DNA. Low copy number samples

(containing less than50picogramsofDNA)aswell as lowquality,degraded samples require

highlyefficientcollection,extraction,andamplificationprocedures.Thesesamplesareseenin

avarietyofforensicevidenceincludingtouchevidenceandagedsamples.

PCR inhibitors are another challenge and must be eliminated before downstream

applications can be performed. Common inhibitors are indigo dyes from denim, heme from



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blood,humicacidfoundinplantsandsoil,andcollagenfoundinvarioustissues.Themajority

of these inhibitors are effectively eliminated using silica‐based DNA extraction methods or

additionalpurificationwithchargeorsizeexclusioncolumns. Thepresenceof inhibitorscan

bedetectedbyperformingPCRwithinternalpositivecontrols.Ifpresent,someinhibitorscan

beneutralizedbyvarioustreatmentsincludingsodiumhydroxidewashesorfurtherpurification

withMilliporeMicroconYM®columns.

IntegrationofEvidenceCollectionwithPost‐CollectionProcessing.Theneedtoreconcile

the“realworld”requirementsofsamplecollectionwiththemicrofluidicrequirementsofafully

integrated microfluidic DNA processing biochip can be referred to as the “macro‐to‐micro

interface”orthe“world‐to‐chipinterface”(Fredrickson2004).Muchofthereportedresearch

onaddressingthisinterfaceisfocusedonresolvingthemismatchbetweenthemacrofluidicand

microfluidic volumetric requirements, but little of no research concerning the reconciling of

specific forensic sampling requirements and formats with microfluidic devices has been

reported.

The(non‐forensic)volumetricmismatchhasbeencommerciallyaddressedbyAgilentin

theBioanalyzer2100bytheuseofacapillarytoaspiratesamplesfromamicrotiterplatetoa

chip for enzyme assays (Lin 2003). Similarly, Gyros has developed a capillary dispenser for a

LabCDsystemwheresamplesareaspiratedfromawellplateintoadispensingnozzleandthen

directedupwardsontoarotatingdevice(Jesson2003).Thesedevices,however,donotaddress

the format incompatibility of collected forensic samples—particularly on the commonly used

collectiondevicesbasedonswabs.



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D. RequirementsofaSampleCollectionSystem

Sample collection and initial sample processing are critical to the development of a fully

integrated microfluidic system for STR typing. These steps are macrofluidic and must be

conducted such that theendproduct—DNA in solution—canbe transferred toamicrofluidic

biochip.TheevidencecollectiondeviceandSmartCartridgedevelopedaspartofthisresearch

addresstheformatandvolumetricmismatchthatexistsbetween“realworld”forensicsamples

that are collected and requirements for analysis by microfluidic devices, a mismatch that

precludes direct insertion of biological samples collected by validated forensic collection

methods directly into a microfluidic biochip. This research takes advantage of a series of

forensic advances in sample collection, elution, and processing of difficult samples. These

forensic advanceswere combinedwithBode’sexperiencewith sample collectionandelution

andNetBio’sexperiencewithmicrofluidicmanipulationtodevelopasamplecollectiondevice

andSmartCartridgecompatiblewiththemicrofluidicbiochiprequiredforafullyintegratedSTR

typingsystem.

Thecollectionsystemwasdesignedtopossessthefollowingproperties:

Ease of use—The evidence collection system must be capable of operation by law

enforcementagentswithminimaltraininginmolecularbiologicalorchemicalanalysisandmust

protectagainst inter‐andintra‐runsample‐to‐samplecontamination. Thesystemwillbepre‐

filledwithallreagentstoreducelaborrequiredtooperatethesystem.

• SampleTypes—Thesystemmustacceptavarietyofsampletypes.Forthisproject,we

havefocusedonliquidanddriedblood,buccalcells,epithelialcells,andsalivasamples.



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• Sensitivity—The systemmust be capable of accepting and processing sampleswith a

highdynamicrangeofcellcounts.

• Compatibility—Both the evidence collection device and Smart Cartridge must be

compatiblewithallrequiredreagents.

• Integration—Allevidencecollectionmatricesandsampleprocessingreagentsmustbe

compatiblewithtransferusingmicrofluidicprinciples.

• Timetoanswer—Thesampleprocessingproceduremusttakenomorethan5minutes.

IV. ResearchDesignandMethods

A. OverviewofResearchDesign

Thegoalofthisresearchwastodevelopasamplecollectionandprocessingsystemconsisting

ofanevidencecollectiondeviceandasampleprocessingdevice termeda“SmartCartridge.”

TheSmartCartridgewilllyseandsolubilizetheinputsample,purifyDNA,andconcentratethe

purifiedDNA. The concentratedDNAwill thenbe transferredautomatically toNetBio’s fully

integrated microfluidic biochip for further DNA purification, DNA quantitation, STR

amplification, and separation and detection. Figures 1 and 2 show schematics of the entire

process,fromevidencecollectiontogenerationofanSTRprofile.

Thespecificobjectivesofthisresearchwereto:

Develop an evidence collection device that provides for ease of biological sample

collection, protection, and documentation. This includes the evaluation and selection of a

collectionmatrix thatprovidesefficiencyofbiological samplecollectionoverawide rangeof

sampletypesandcompatibilitywithsubsequentsampleprocessingincludingsamplelysis,DNA

solubilization, and concentration in the Smart Cartridge, and also subsequent downstream



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sampleprocessing in the integratedmicrofluidicbiochip.Theevidencecollectiondevicemust

protectthesampleforstorageandtransportandpreventsample‐to‐samplecontamination.

Develop a Smart Cartridge that accepts the evidence collection device, performs

processing steps, and transfers the resulting DNA solution to the microfluidic biochip. This

sample processing cartridge will incorporate sample lysis, DNA solubilization, and

concentration.Appropriatemechanicalandfluidicinterfacesforcouplingtoboththeevidence

collectiondeviceandthebiochipwillalsobeincorporated.

B. MaterialsandMethods

1. MockCaseworkandReferenceSamples.

Buccal cell samples were obtained bymoving the swabs up and down on the inside

cheekofahumansubject.

FreshwholebloodcontainingEDTAasanticoagulantwasobtainedonice.

Driedbloodsamplesonswabwerepreparedbyallowingthebloodtodryovernight.

Salivawascollectedbyexpectoratingintoa50mlfalcontube.

Epithelial cell sampleswere collected by rubbing the swab heads on the palm and in

betweenthefingersofahumansubject.

A set of reference and mock casework samples was also prepared by Bode. These

samplesconsistedobuccal,blood,driedblood,saliva,andepithelialcells thatwerecollected

anddriedonSecurSwabs.

2. VortexExtractionofDNAfromSwabs.

Swabswereplacedina2mLmicrocentrifugetube.Theswabheadswereseparatedfromthe

shaft by cutting them off with scissors. 500 µL of NetBio lysis solution was added to the



26

microcentrifugetubeandthetubewasvortexedfor5seconds.Theswabheadsweremanually

removedusingapairofcleantweezersanddiscarded.

3. MechanicalAgitationforExtractionofDNAfromSwabs.

A tube containing lysis solutionwas placed in a custombuiltmechanical agitation system.A

SecurSwabwasinsertedintotheagitationsystemandattachedtothesystembytheswabcap.

Aminiaturizedmotorwasused togeneratevigorousvibrationsand this vibrationalenergy is

coupled to the swab tip through the cap and shaft. Mechanical agitation was applied

immediatelyafterinsertionintothesystem.

4. MicrofluidicBiochipPurification.

ThemicrofluidicbiochipcontainsapurificationfilterforDNAbindingsealedwithinachamber.

Twomicrofluidicchannelsleadtothechamber,onetoallowflowofreagentstothefilterand

theother to remove reagents from the filter. The lysis solutionmixturewas loaded into the

input port and pneumatically driven through the purification filter. This was followed by

pneumaticallydrivingwashbufferthroughthefilter.BoundDNAwaselutedfromthefilterby

pneumaticallydrivingelutionbufferthroughthefilter.

5. Automated Microfluidic Extraction and Purification of DNA from Swabs in

SmartCartridge.

The Smart Cartridge is comprised of reagent chambers for holding preloaded solutions and

processchambers.OnechamberisusedtoholdthecottonswabwiththeDNAsample;fourof

the chambers are prefilled with purification reagents. The final two chambers are used for

holding solutions during the process. The SC biochip is comprised of a network of channels,



27

chambers, flowcontrolelements,andapurification filter.Anautomatedscriptpneumatically

manipulatessolutionswithintheSCtoextractDNAfromacottonswabthatisinserted.

6. STRAmplificationReaction.

MultiplexPCRreactionswereperformedwiththeAmpFℓSTR®Identifiler®PCRAmplificationKit

(AppliedBiosystems,FosterCity,CA).The7µLPCRreactionandcyclingprotocolwasprepared

as described in (Giese 2009). Amplification of 16‐sample in the microfluidic biochip was

completed in approximately 17minutes and the amplifiedproductsweremanually retrieved

fromtheindividual.

7. STRseparationanddetectioninstrumentation

Amplified products were separated and detected using Genebench‐FX. This instrument was

developedandoptimizedspecificallyforSTRanalysis.

V. DisseminationStrategy

Theresultsofthisresearchhavebeendisseminatedasfollows:

Presentations. Thisworkhasbeenpresentedinanoralpresentationentitled“Sample

CollectionSystemforDNAAnalysisofForensicEvidence”attheAmericanAcademyofForensics

Scientists62ndAnnualScientificMeetinginSeattle,WAonFeb22‐27,2010.

Duringandafterthecourseoftheproposedwork,wehavediscussedourprogressand

askedforsuggestionsandcommentsfromseveralforensicscientists.Wehavemaintainedand

expanded contact with the NFSTC and several state crime labs. Our hope is to continue

developing a close relationshipwith these groups to ensure that our development goals are

consistentwiththeneedsoftheforensiccommunity.



28

VI. ImplicationsforCriminalJusticePolicyandPractice

The availability of an STR typing instrument that combines DNA extraction and purification,

amplification, and separation into a single, easy to operate instrument would represent a

substantialadvanceinforensicDNAanalysis.Significantburdensinsettingupandoperatinga

forensic DNA analysis laboratory are the costs of dedicated rooms to prevent PCR

contamination,automatingtheprocedures(eitherthroughroboticsordedicatedtechnicians),

and validating and re‐validating individual instruments and the entire series of laboratory

processes. A fully integrated instrument has the potential to be faster,more sensitive, less

susceptible to contamination, less costly, and less labor‐intensive than currently available

technologies.

Indevelopinga fully integrated system for STR typing, evidence collectionand

initial sample processing are critical steps, and, as such,must be incorporated into any fully

integratedmicrofluidicSTRtypingsystem.Thisresearchhasresulted inthedevelopmentofa

practical system for DNA analysis of forensic evidence, a system that is faster and performs

better than currently available technologies. The research represents an important step

towardsmakingfullyintegratedforensicDNAanalysisareality.

Finally, a fully integrated microfluidic STR typing instrument would offer forensic

scientistsnewcapabilitiesnotpossiblewithconventionalcapillaryinstrumentation.NetBiohas

alreadyruggedizedGenebench‐FXanddemonstratedthatitwithstandstherigorsoftransport

to and operation at the crime scene. Developing an evidence collection device and Smart

Cartridge capableof collecting sample andprocessingDNA froma varietyof biological fluids



29

associated with a variety of evidence will also contribute towards producing reliable DNA

profiles in a cost‐effective manner at the crime scene. The use of a field portable, fully‐

integratedDNAanalysis instrumentwill allow rapid generationof aDNAprofilewhich could

directlyaffecttheapprehensionofasuspectandtherebyreducecrimeandimprovethepublic

safetywhiledecreasingthetimeandcostsofcriminalinvestigations.

VII. ResultsandDiscussions

A. Evidencecollectionmedia

TheobjectiveofthisworkwastodeterminethebestmatrixtocollectandextractDNAfroma

varietyof cells associatedwithdifferentPCR inhibitors, varietyof surfaces, and fromvarying

cellnumbers.Aseriesofcollectionmatricesconsistingofnatural fiber (cotton)andsynthetic

matrices(modifiedcellulose,foam,Nylon,PolyesterandRayon)wereselectedforevaluation.

Fourbuccalsampleswerecollectedfromeachdonorwitheachofthecollectionmatrices.The

collection matrices were assessed based on DNA collection and extraction efficiency, lysate

retentionvolume,anduseinforensicapplications.

DNAcollectionandextractionefficiency.DNAwasextractedandpurifiedfromeachof

the 112 samples using a set of reagents thatwere optimized for purification of buccal swab

samples, compatibility with biochip DNA purification, and minimal process time. DNA yield

measuredbyUVanalysis showedthatawide rangeofDNA (300‐1900ng)wasextractedper

swabwithhigh inter‐ and intra‐ donor variation. The average recoveredDNAacross showed

thatallswabtypesperformedsimilarly(Figure16).



30

Figure16.DNArecoveredfromvariousswabmatrices.

Use in forensic applications. A survey of swab collection matrices used in forensic

applicationssuggestedthatcottonwasmostcommonlyusedforcrimescenesamplecollection

andforreferencesamplecollection.

Cotton was the matrix selected for incorporation into the sample collection device

becauseofitswidespreadacceptanceandusewithintheforensicscommunityandcomparable

extractionefficiencyrelativetoothertestedcollectionmatrices.

Commerciallyavailableswabswithvariousswabheadconfigurationswereassessedfor

useinthefullyintegratedsystem.Easeofuseforbuccalcellcollectionwasevaluatedbyhaving

eachdonorswabtheinsideofthecheekwiththecandidateswab.Furthermore,eachcandidate

swabwasalsousedtocollectmockcrimescenesampleswiththecandidateswabs.Subjective

feedback frombuccal cell donorsand crime scene collectorswereevaluated toestablish the



31

SecurSwab (Bode) as the easiest, safest, and most convenient for use; the SecurSwab was

designedforcollectionofDNAsamplesorforensicevidenceandiseasiertoopen,holdanduse.

Thereproducibilityoftheevidencecollectorwasevaluatedbycollecting4buccalswab

samplesfromeachofnineteendonors.Twoswabswerecollectedfromtheleftcheekandtwo

from the right cheek. DNA from each swab was extracted and purified. Purified DNA was

quantifiedbyUVspectrometry.Data inFigure17showsthatDNAyieldedfromthe76swabs

rangefrom200ngto1850ng,andthattheswabtoswabvariation(%CV)forthe76samples

was42%.Thecoefficientofvariation (%CV)withineachdonorranges from20to65%.These

results show that DNA collected from buccal swabbing is highly variable, however, the

minimum amount that is collected by the selected evidence collection device is more than

sufficienttogeneratethe1ngofDNArequireforSTRanalysis.



32

Figure17.DNAyieldfrombuccalswabscollectedacross19donors.

Figure 18 shows the results of an STR analysis generated by amplifying 1 ng of DNA

templateandtheAmpFLSTRProfilerPrimerset,andseparatinganddetectingwithGenebench.

TemplateDNAwascollectedfrombuccalcellscollectedbytheevidencecollectiondeviceand

extractedandpurifiedwithNetBioreagentsfollowingvortexextraction.ThefullSTRprofilethat

isdemonstratedconfirmsthecompatibilityof theevidencecollectiondeviceand thereagent

setforSTRanalysis.

The performance of NetBio’s microfluidic purification biochip was evaluated by

generating sets of lysates from swabs, by vortex extraction, and purifying with NetBio’s

Microfluidic Biochip Purification protocol. The purified DNA was quantified and 1 ng of the

purifiedsolutionsubjected toamplification.The full STRprofile (Figure19)demonstrates the

compatibilityofthereagentsetwithmicrofluidicbiochippurifications.

B. ChemicalLysis

Biologicalmaterialcollectedwiththeevidencecollectoris lysedandDNAissolubilized

within the Smart Cartridge. The solubilized DNA is then microfluidically transported for

subsequentDNApurification.It isimportanttomaximizethequantityofDNArecoveredfrom

the sample collection device, particularly in casework sampleswhere the amount of sample

collectedmaybelimited.



33

Figure18.STRprofileofDNApurifiedfrombuccalswabsampleswithNetBio’soptimizedreagentsetfollowingvortexextraction.

Figure19.STRprofileofDNAmicrofluidicallypurifiedinbiochipfrombuccalswabsampleswithNetBio’soptimizedreagentstefollowingvortexextraction.



34

Initial work on cell lysis strongly suggested that chaotropic salts are effective lysis

reagents and are compatible with microfluidic biochips. Optimization of the chemical lysis

reagent components was performed to maximize DNA efficiency for sample types including

blood, buccal, touch, and saliva samples, and to minimize process time. The optimization

includes a systematic variation of the lysis reagent components, wash solution components,

andelutionbuffercomponents.Tominimizethenumberofiterations,anexperimentalmatrix

wasdeveloped to determine the effect of each componentonDNAyield andpurity, and an

optimal reagent set. Three formulations (NetBioA,B andC)wereevaluatedby collecting12

buccalswabsamplesfromthesamedonorandpurifyingtheDNAfromthebuccalswabswith

each of the formulations. The DNA yielded was quantified by spectrometry shows that

formulationCgenerates thehighestaverageDNAyieldof900ngcomparedwith850ngand

650ngforformulationsBandArespectively.

C. SmartCartridgeProcessStep2:SelectionofSilicaFiberMembrane

DNAConcentrationandpurification

Thebindingandelutionpropertiesofchaotropicsalt/silicamediasystemsallowDNAto

be concentrated from relatively dilute lysates. An added advantage of using the bind/elute

method for DNA concentration is the opportunity towash theDNAwhile it is bound to the

membrane,allowingfortheremovalofparticulatesandcrudecelldebrispriortoelution.Silica

basedbind/elutemediaareavailable in several formats including silica fibermembranesand

silicabeads.Useofthemembraneformatrequiresthemembranetobepositionedatafixed

site of the Smart Cartridge,with the attendant advantages of lowermanufacturing cost and

ease of containment of the silica media. The bead‐based format provides the flexibility of



35

movingthebeadsto leavebehindthedebrisassociatedwiththecollectedsamplesand lysed

cells. This format is relatively costly to implement, and the beads are somewhat difficult to

contain.Accordingly,performanceevaluationshavebeenconductedonmembranebasedsilica

fibermedia.

D. SmartCartridgeDevelopment

An initial Smart Cartridge was designed and fabricated based on the optimized

conditionsdiscussedintheprevioussections.Thisplatformformedthebasisfortheevaluation

andoptimizationofthethreeprocessstepsinseries.TheinitialSmartCartridgewasdesigned

withthefollowingfeatures:

• Flexibility – ability to rapidly implementmodifications toboth the flowconfigurations

andprotocols

• Linked processing chambers – the lysis and DNA concentration are in direct

communication.

• Optical imaging – the device functions with optical cameras to allow the research

monitoring of reactions within each chamber and fluid flow within and between

chambers.

• Fluidicdrive–pneumaticandfluidicpumpsarecoupledtotheSCtodriveprocessfluids.

• Computer‐controlled – the Smart Cartridge is computer‐controlled to allow the

automatedexecutionofaprocessbyscript

A single sample smart cartridge design and associated extraction and concentration

processflowshasbeendeveloped.ArenderingofasingleSC(Figure20)showsamacrofluidic

componentforacceptingaBodeSecurSwab,storingreagents,andprocessing.



36

Figure20.Renderingofasinglesamplesmartcartridge.

E. TandemEvaluationandOptimizationoftheThreeProcessSteps

TheSCperformancewascharacterizedusingbuccalswabs.DNAfromtheprocesswas

quantifiedbyUVabsorbanceshowsthatbetween200–1100ngofDNAisyieldedfromthe16

swab samples (Figure 21). This yield of DNA is comparable to manually processing and

demonstratesthesuccessofautomatedsmartcartridgeprocess.



37

Figure21.DNAextractionperformanceoftheextractioncomponentoftheSC.

F. TestingoftheEvidenceCollectionDeviceandSmartCartridge

Theabilityof thecompletedevidencecollectiondeviceandSmartCartridge tocollect

samplesandtoperformthethreeprocessstepswastested.Aseriesofmockcaseworksamples

werecollectedandprocessedasfollows:

Whole blood. Two evidence collectors were used to swab the blood directly from a

ceramicsurface.OneevidencecollectorwasprocessedbyautomatedscriptintheSCandthe

other was processed following a control Qiagen protocol. DNA from the SC process was

amplifiedandseparatedanddetectedonGenebench.Figure22showsafullSTRprofileofthe

SC processed sample. This profile is similar to that generatedwith the control protocol and

demonstratesthecompatibilityofthesamplecollectionsystemforwholebloodsamples.

Driedblood.TwoevidencecollectorswerewetwithDIwaterandusedtoswabthedried

bloodfromthesurface.Oneevidencecollectorwasprocessedbyautomatedscript intheSC,



38

andtheotherevidencecollectorwasprocessedfollowingtheQiagenprotocol.DNAfromthe

SCprocessandtheQiagenprocesswasamplifiedandseparatedanddetectedonGenebench.

Figure 23 shows a full STR profile of the SC processed sample. This profile is similar to that

generated with the control protocol and demonstrates the compatibility of the sample

collectionsystemfordriedbloodsamples.

Figure22.STRprofileofDNAextractedandpurifiedintheSCfromwholeblood.



39

Figure23.STRprofileofDNAextractedandpurifiedintheSCfromdriedblood.

Salivasamples.Pooledsalivasampleswerecollectedandspottedonaceramicsurface.

Twoevidencecollectorswereusedtoswabthesalivafromthesurface.Theevidencecollectors

werethensecuredintheircollectiontubesovernight.Oneevidencecollectorwasprocessedby

automated script in the SC, and the other evidence collector was processed following the

Qiagen protocol to serve as controls. DNA from the SC process and theQiagen processwas


SCprocessedsample.

Buccalsamples.Buccalsampleswerecollectedbyswabbingtheinsideofthecheekand

then secured in their collection tubes overnight. One evidence collector was processed by

automated script in the SC, and the other evidence collector was processed following the



40

Qiagen protocol to serve as controls. DNA from the SC process and theQiagen processwas


SCprocessedsample.

Figure24.STRprofileofDNAextractedandpurifiedintheSCfromsaliva.



41

Figure25.STRprofileofDNAextractedandpurifiedintheSCfrombuccalcells.

VIII. Conclusions

This report presents work demonstrating successful completion of all technical

milestonesofthisresearchproject.

An evidence collection device has been developed for ease of biological sample

collection,protection,anddocumentation.Theevaluationofvarietysamplecollectionmatrices

including cotton, modified cellulose, foam, nylon, polyester and rayon based on collection

efficiency and current use in forensics applications resulted in the selection of cotton.

Commerciallyavailableswabswithvariousswabheadconfigurationswereassessedforeaseof

useandresultedintheselectionoftheBodeSecurSwabforincorporationintothesystem.

A Smart Cartridge was developed to accept the evidence collection device, performs

processing steps, and transfers the resulting DNA solution to the microfluidic biochip. The



42

optimization of a lysis and concentration reagent set, and incorporation of mechanical lysis

resultedintherapidandefficientlysisofbuccalcellsamplestoyield200–1400ngofDNA.A

Smart Cartridge was fabricated by initially designing and fabricating the macrofluidic and

microfluidiccomponentsoftheSC,andprocessingofbuccalsamplesdemonstratedthatDNAof

between200–1100ngofDNAisyieldedbytheautomatedSCprocess.

The system (evidence collector and SC) was tested by collecting mock crime scene

samplesincludingwholeblood,diedblood,saliva,andepithelialcellsampleswiththeevidence

collectorandprocessingwiththeSCbyautomatedscript.STRprofilesofDNAgeneratedbythe

systemweresimilartothoseproducedbyconventionalextractionprotocols.

Theresultsasawholedemonstratethesuccessfuldevelopmentofanevidencecollector

and SmartCartridge capableofpurifyingDNA froma varietyof sample types and substrates

relevanttotheforensicssciencescommunity.Thesuccessfuldevelopmentofthismodulealso

represents the completion of another critical step towards the implementation of a fully

integratedinstrumentthatwillgenerateanSTRprofilein45minutesfromsampleintroduction

withminimal operating requirements. The integrated instrument will process 16 samples in

parallelanddramaticallyreducethecosts(includinglabor,space,andvalidation)ofsettingup

andoperatingaDNAlab.TheevidencecollectiondeviceandSmartCartridgearedesignedsuch

that the systemwill be easy to operate and compatible with both forensic andmicrofluidic

requirements.

IX. References

Bienvenue,J.etal.(2010).AnIntegratedMicrofluidicDeviceforDNAPurificationand

PCRAmplificationofSTRfragments.ForensicSciIntGenet4(3):178‐86.



43

Fredrickson, C. K. and Z. H. Fan (2004). "Macro‐to‐micro interfaces for microfluidic

devices."LabChip4(6):526‐33.

Giese, H., R. Lam, et al. (2009). "Fast multiplexed polymerase chain reaction for

conventionalandmicrofluidicshorttandemrepeatanalysis."JForensicSci54(6):1287‐96.

Hopwood, A. et al. (2010). Integrated Microfluidic System for Rapid Forensic DNA

Analysis:SampleCollectiontoDNAProfile.AnalChem2010,82:6991‐9.

Jesson,G.etal.(2003).MicroTotalAnalysisSystems,ed.M.A.Northrup,K.F.Jensen

andD.J.Harrison,pp.155–158.

Lauk,C.andJ.Schaaf(2007)."ANewApproachfortheExtractionofDNAfromPostage

Stamps."ForensicScienceCommunications9(1).

Lazzarino,M.F.,A.ColussiandM.M.Lojo (2008)."DNARecovery fromSemenSwabs

withtheDNAIQSystem."ForensicScienceCommunications10(1).

Lee,H.C.andC.Ladd(2001)."Preservationandcollectionofbiologicalevidence."Croat

MedJ42(3):225‐8.

Lee,H.C.,C.Ladd,C.A.ScherczingerandM.T.Bourke(1998)."Forensicapplicationsof

DNA typing:part2: collectionandpreservationofDNAevidence."AmJForensicMedPathol

19(1):10‐8.



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Leemans,P.(2006)."EvaluationandmethodologyfortheisolationandanalysisofLCN‐

DNAbeforeandafterdactyloscopicenhancementoffingerprints."IntCongressSer1288:583‐

585.

Lin, Y.W.,M. J. Huang andH. T. Chang (2003). "Analysis of double‐strandedDNA by

microchip capillary electrophoresis using polymer solutions containing gold nanoparticles." J

ChromatogrA1014(1‐2):47‐55.

Norris,J.V.,K.Manning,S.J.Linke,J.P.FerranceandJ.P.Landers(2007)."Expedited,

chemicallyenhancedspermcellrecoveryfromcottonswabsforrapekitanalysis."JForensicSci

52(4):800‐5.

Voorhees,J.C.,J.P.FerranceandJ.P.Landers(2006)."Enhancedelutionofspermfrom

cottonswabsviaenzymaticdigestionforrapekitanalysis."JForensicSci51(3):574‐9.


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