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AppliedGeneticsRapid Forensic DNA Typing:Protocols and InstrumentationPeter M. ValloneDNA Biometrics Project Leader, Applied [email protected] 2012 MeetingGaithersburg, MDNovember 28, 2012

Forensic STR TypingAppliedGeneticsCollectionUsually 1-2 day process (a minimum of 8 hours)Specimen Storage1.5hoursExtraction1.5hoursQuantitationBlood Stain Buccal swabMultiplex PCRSample Collection& StorageDatabaseDNAQuantitation3.5hoursSTR TypingInterpretationof ResultsDNAExtractionStatistics CalculatedDNA Database searchPaternity testReference sampleMultiplex PCR AmplificationDNA separation and sizingStorage & SearchingCalculation ofMatch ProbabilityApplied Use ofInformation1.5hoursSTR TypingInterpretation of Results

AppliedGeneticsWhat is Rapid Forensic DNA Typingor Rapid DNA (R-DNA)? Generating a STR profile in minutes vs hours– 90 minutes versus 6-8 hours– Single-source reference samples (not casework) Non-integrated– Laboratory based (existing equipment)– Specially trained analysts– Robotics, fast PCR, direct PCR, quick extraction, etc Integrated approach– Fully integrated microfluidic platform– ‘Swab in – answer out’– Non-expert user

AppliedGeneticsBenefits and Applications Faster sample-to-answer turnaround times Increased throughput for databasing labs Impact of Integrated R-DNA platforms–––––Booking stations, investigative leadsRapid intelligence, field testingMass fatality, disaster victim investigationKinship determination, immigration, border securityInterest in R-DNA by FBI, DHS, DoD

AppliedGeneticsImportant Questions Can a quality result be obtained with rapidtechniques?– Uphold DNA as the gold standard for human identification– Reference/database or casework samples?– How do we validate rapid techniques and instruments? Robustness Reliability Reproducibility Concordance ‘the correct answer’SensitivityContamination, mixturesStutter, peak height balance, artifacts

AppliedGeneticsOngoing Projects that Support R-DNA Non-integrated––––Developing rapid PCR protocols for STR kitsFaster thermal cyclers and DNA polymerasesDirect PCR kit evaluationRapid typing workflows (Sampling through Profile) Integrated approach– Performance assessment of prototype R-DNAinstruments– Inter-laboratory study

AppliedGeneticsRapid PCR Protocols Reducing the time required for PCR– 3 hours down to sub-30 minute Accomplish this by optimizing conditions for:– Faster DNA polymerases– Faster thermal cyclers

AppliedGeneticsPCR Thermal CyclersCycling TimeCycler(min)GeneAmp 970036Mastercycler Pro S19Rotor-Gene Q36SmartCycler22Philisa17Piko30SpeedCycler222Palm PCR1795oC for 1 min95oC for 5s28x 58oC for 10s72oC for 10s72oC 1 minPeter Vallone: Green Mountain DNA Conference (Burlington, VT), August 3, 2012, "Development ofProtocols for Rapid Amplification of STR Typing Kits: The Use of 'Non-Standard' Thermal Cyclers"

AppliedGeneticsDNA Polymerases AmpliTaq Gold is typically used– Heat activated (avoid nonspecific PCR products)SpeedSTAR HS DNAPolymerase– Extension times of 100 bp/sare possible (compared to 20bp/s for other polymerases)– Hot-start formulation isantibody mediatedQiagen– QIAGEN Fast Cycling PCR Kit New England Biolabs/Finnzymes– Phusion and Phire DNAPolymerases KAPA Biosystems– KAPA2G Fast PCR Kits Biotium– CheetahTM Taq Fermentas– PyroStart Master Mix EMD Millipore– KOD DNA Polymerse

1 ng of DNA templateAppliedGeneticsGeneAmp 9700 31 min PCR

1 ng of DNA templateAppliedGeneticsPhilisa 17 min PCR

Benefits of Direct PCRAppliedGenetics Sample set-up convenience: ‘punch and go’Amplify unpurified DNA - skip extraction and quantitationAmenable to automationApplications: offender DNA database samples, paternitysamples, casework reference Separation &DetectionCollectionDirect PCRAmplificationDataInterpretation

PowerPlex 18D: 1.2 mm Blood punch off FTA paperAppliedGenetics90 minute PCR cycling time

AppliedGeneticsExample Rapid Typing WorkflowNon-Integrated (Lab) Settingsingle source reference samplesDNA extractionRapid PCRCE separationand detection Extraction: Prep-N-Go Buffer PCR: Rapid Identifiler (Philisa cycler) Separations: 8 capillary 3500 Genetic AnalyzerDirect PCRCE separationand detection PP18D (9700 cycler) Separations: 8 capillary 3500 Genetic Analyzer8 unique samples were typed in parallel

AppliedGeneticsExtraction rPCR Separation/DetectionTotal time from swab to answer: 57 min 42 secIncludes set up times

AppliedGeneticsDirect PCR Separation/DetectionTotal time from swab to answer: 2 hours 16 min 45 secIncludes set up timesErica Butts poster at the 23rd International Symposiumon Human Identification (ISHI) meeting (Nashville, TN),October 16-17, 2012, "Rapid DNA testing approaches forreference samples" Lecture Room BWhatman EasiCollectTM

AppliedGeneticsIntegrated Approach to Rapid DNAFully automated (hands free) process of developing a CODISCore STR profile from a reference sample buccal swabBuccal swabR-DNA InstrumentDNA extractionPCRSeparation and DetectionAllele 1,15}{9,11}{19,22}{X,Y}

AppliedGeneticsDevelopers of R-DNA Instrumentation IntegenX NetBio ZyGem/Lockheed Martin Univ of Az

AppliedGeneticsPerformance Testing Goals Testing of R-DNA platforms for baselineperformance of Robustness, Reliability, andReproducibility Type similar sample sets on multiple instrumentsand from multiple vendors Results will help guide platform improvementsand additional testingCarry this out through aninter-laboratory study

AppliedGeneticsNIST Inter-laboratory Test Samples 50 samples (buccal swabs) will be provided toeach participant– Five replicates of 10 anonymous individuals– NIST IRB approval– Each individual typed at NIST (PowerPlex 16 HS)

What will this data provide?High levelAppliedGenetics Is the correct profile obtained? Typing successTotal Runs– Per lane, chip, overall Incorrect profilesPartial profilesAllele drop outContaminationGeneral operational issues– Instrument/chip failures– Hardware and softwareTotal LanesLanes with correct CODIS 13% CODIS 13 loci442209041%Lanes with correct PP16% PP16 loci8237%Failed lanes (CODIS 13)Failed chip eq13026

What will this data provide?Detailed-expert user; developerAppliedGenetics Electropherogram characteristics––––Signal intensityPeak balance (inter- and intra locus)Stutter, PCR artifacts, adenylationSizing precision of peaks Manual versus automated allele calls– Confirm optimal software allelecalling parametersstutter33.6%Peak balance

AppliedGeneticsInter-laboratory Testing Results Provide participants and sponsor with data and feedback Each participant and will receive their specific performance feedback The sponsor (FBI) will get a cumulative report for disseminationData and results will assist ongoing developmental validationstudies and other decisions in the adoption of R-DNALab 1Lab 2Lab 2Lab 3Lab 4Lab 5(vendor)Testing R-DNA Instrument ANISTAnalyzedResultsResults disseminatedSend out samplesNISTInterlaboratorySamplesData returned to NISTLab 1Lab 3Lab 4Lab 5(vendor)CumulativeReport toSponsor (FBI)

AcknowledgmentsAppliedGeneticsFunding from theNational Institute ofJustice (NIJ) throughNIST Office of LawEnforcement StandardsFunding from the FBIBiometrics Center ofExcellence ‘DNA as aBiometric Tool’Erica ButtsInitial rapid PCR workContact Info:[email protected] Science and Technology (Chris Miles)NIST Disclaimer: Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures ascompletely as possible. In no case does such identification imply a recommendation or it imply that any of the materials, instruments orequipment identified are necessarily the best available for the purpose.Points of view are those of the presenters and do not necessarily represent the official position of the National Institute of Standards andTechnology or the U.S. Department of Justice.

AppliedGeneticsThank you for your attention!

Protocols for Rapid Amplification of STR Typing Kits: The Use of 'Non-Standard' Thermal Cyclers" Cycler Cycling Time (min) GeneAmp 9700 36 Mastercycler Pro S 19 Rotor-Gene Q 36 SmartCycler 22 . –PyroStart Master Mix EMD Millipore –KOD DNA Polymerse . Applied Genetics GeneAmp 97