Rapid DNA Analysis DNA: the Ultimate Biometric? Advances in Molecular Processing and Analysis

Transcription

1 Rapid DNA Analysis DNA: the Ultimate Biometric? Advances in Molecular Processing and Analysis Presentation at NSF Workshop on Fundamental Research Challenges for Trustworthy Biometrics Dr. Joan Bienvenue Chief Scientist, Program Manager, Lockheed Martin and Dr. James P. Landers University of Virginia and ZyGEM-MicroLab Diagnostics Dr. Joan Bienvenue 1

2 OUTLINE Future Directions for Biometrics DNA Microfluidics Where are we at? Where do we need to be? Beyond STRs: Next Generation Technological Advances SNPs NextGen sequencing New Technology, New Problems? Policy, ethics, legal, security concerns Dr. Joan Bienvenue 2

3 Biometric Modalities smell DNA: The Ultimate Biometric? Dr. Joan Bienvenue 3

4 Challenges with DNA As A Biometric Different applications, different specs Portability Training of end users LCN Casework Evidence quality Interpretation of results Speed of analysis Court acceptance Dr. Joan Bienvenue 4

5 Our Motivation Current Approach 9 hrs, 50 min (average) 8 hrs, 15 min (rush) Cell Lysis/ DNA Extraction gdna Quantification Dilution PCR Amplification Detection 1hr 45 min 2 hr 20 min 15 min 3 hr 30 min 2 hr Qiagen EZ1 ABI 7000 RT-PCR Tecan Genesis ABI 9700 Multimek ABI 3130 LM-ZyGEM/MicroLab Approach Alpha Device INTEGRATED DEVICE Step 1: Sample Collection Step 2: Liquid Extraction Step 3: Polymerase Chain Reaction Step 4: Electrophoresis Separation/Detection Step 5: Analysis Ruggedized instrument at TRL 6-7 Easily portable Microfluidics Approach Inexpensive Portable Robust Fast Dr. Joan Bienvenue 5

6 RapI.D. STR Chip Micro-Analysis Microvolume Analysis: Ultrafast processing Open channel architecture Reduced reagent needs Closed analysis environment Small analytical footprint Microfluidic interconnects Integrated Instrument: Sample in-answer out Turn-key operation Miniaturizable footprint Portable Automated Unskilled personnel Dr. Joan Bienvenue 6

7 Anticipated LM-ZM Instrument Ruggedized instrument at TRL 6+ Modular Component design to support maintainability Easily portable Removable PC 4, 8, 12, 16 channel cartridges containing reagents Plug and Play concept Portable (< 40 lbs) Ultra compact Dr. Joan Bienvenue 7

8 Micro-Total Analysis STR Profiling in <45 min? +5 min for fluidic pumping, etc. <4 min Extraction Separation SPE Inlet 8 min <2 min <20 min Amplification Injection STR Profile DNA Purification DNA Amplification Injection Separation/D etection Multiplex amplification High resolution separation Fluidic integration key Dr. Joan Bienvenue 8

9 NSF Workshop on Fundamental Research Challenges for Trustworthy Biometrics 2010 Rapid Microfluidic DNA Analysis A Reality Multiple competitors, research born out of high quality university effort Close ties to biometric and forensic analysis communities Small companies working together with universities, larger corporations to bring the best in technology and innovation to these analyses Rapid, portable analysis is possible Dr. Joan Bienvenue 9

10 Motivation Expedite and Miniaturize 9 hrs, 50 min (average) 8 hrs, 15 min (rush) Cell Lysis/ DNA Extraction gdna Quantification Dilution PCR Amplification Detection 1hr 45 min 2 hr 20 min 15 min 3 hr 30 min 2 hr Qiagen EZ1 ABI 7000 RT-PCR Tecan Genesis ABI 9700 Multimek ABI 3130 Expedite the 3 Major Sample Preparation/ Analysis Steps: Solid Phase Extraction (SPE) Polymerase Chain Reaction (PCR) Separation and Detection Dr. Joan Bienvenue 10

11 Motivation Expedite and Miniaturize 9 hrs, 50 min (average) 8 hrs, 15 min (rush) Cell Lysis/ DNA Extraction gdna Quantification Dilution PCR Amplification Detection 1hr 45 min 2 hr 20 min 15 min 3 hr 30 min 2 hr Qiagen EZ1 ABI 7000 RT-PCR Tecan Genesis ABI 9700 Multimek ABI 3130 Expedite the 3 Major Sample Preparation/Analysis Steps: Solid Phase Extraction (SPE) Polymerase Chain Reaction (PCR) Separation and Detection Dr. Joan Bienvenue 11

12 Generation of High Quality, PCR-Ready DNA min Time reduced from 20 min to <4 min Volume reduced from several hundred L A simple one-step process performed Aids in time reduction 4 min DNA Prep Extraction Separation SPE Inlet 8 min 2 min 40 min Amplification Injection Dr. Joan Bienvenue 12

13 Motivation Expedite and Miniaturize 9 hrs, 50 min (average) 8 hrs, 15 min (rush) Cell Lysis/ DNA Extraction gdna Quantification Dilution PCR Amplification Detection 1hr 45 min 2 hr 20 min 15 min 3 hr 30 min 2 hr Qiagen EZ1 ABI 7000 RT-PCR Tecan Genesis ABI 9700 Multimek ABI 3130 Expedite the 3 Major Sample Preparation/Analysis Steps: Solid Phase Extraction (SPE) Polymerase Chain Reaction (PCR) Separation and Detection Dr. Joan Bienvenue 13

14 Rapid Amplification of STR Loci. Infrared Thermocycling Dr. Joan Bienvenue 14

15 Conventional PCR: IdentiFiler 90 Temperature Total amp. time: ~210 min Time (min) IdentiFiler amplification: 1 ng template DNA 25 µl reaction volume 11 min initial denature, 95 ºC 32 cycles 60 94ºC; 60 59ºC; 60 72ºC 45 min final 72 ºC Conventional amplification with large volume samples is slow Dr. Joan Bienvenue 15

16 Microchip IR-PCR Amplification: IdentiFiler 90 Temperature Total amp. time: ~26 min Modified conditions from Vallone et. al. FSI: Genetics, 2008, 3, Time (min) IdentiFiler amplification: ~1 ng template DNA 0.5 microliter volume 2 min initial denature, 95 ºC 32 cycles 5 94ºC; 10 59ºC; 10 72ºC 1 min final extension 72 ºC IR-PCR amplification with sub-µl volumes is ultrafast Dr. Joan Bienvenue 16

17 Motivation Expedite and Miniaturize 9 hrs, 50 min (average) 8 hrs, 15 min (rush) Cell Lysis/ DNA Extraction gdna Quantification Dilution PCR Amplification Detection 1hr 45 min 2 hr 20 min 15 min 3 hr 30 min 2 hr Qiagen EZ1 ABI 7000 RT-PCR Tecan Genesis ABI 9700 Multimek ABI 3130 Expedite the 3 Major Sample Preparation/Analysis Steps: Solid Phase Extraction (SPE) Polymerase Chain Reaction (PCR) Separation and Detection Dr. Joan Bienvenue 17

18 Peak Resolution in Short Channels High resolution separations in short distances (L eff ) allows for reduced chip footprint and reduced separation times D3S1358 and THO1 9.3/10 9.3/10 resolved in 7cm L eff 8 minute separation time Raw Data Processed Data Dr. Joan Bienvenue 18

19 Motivation Expedite and Miniaturize 9 hrs, 50 min (average) 8 hrs, 15 min (rush) Cell Lysis/ DNA Extraction gdna Quantification Dilution PCR Amplification Detection 1hr 45 min 2 hr 20 min 15 min 3 hr 30 min 2 hr Qiagen EZ1 ABI 7000 RT-PCR Tecan Genesis ABI 9700 Multimek ABI 3130 Expedite the 3 Major Sample Preparation Steps: Solid Phase Extraction (SPE) Polymerase Chain Reaction (PCR) Separation and Detection Dr. Joan Bienvenue 19

20 Integrated DNA STR Profile Dr. Joan Bienvenue 20

21 Automated Allele Calling Dr. Joan Bienvenue 21

22 Portable DNA Analysis Easy, less than 1 hr of training to use Automated allele calling/expert system software Automated profile generation Automated database searches How does this impact the quality, reliability, and acceptability of results? Dr. Joan Bienvenue 22

23 OUTLINE Future Directions for Biometrics DNA Microfluidics Where are we at? Where do we need to be? Beyond STRs: Next Generation Technological Advances SNPs NextGen sequencing New Technology, New Problems? Policy, ethics, legal, security concerns Dr. Joan Bienvenue 23

24 SNPs for Ancestry & Phenotype Inference Useful in cases where a STR profile doesn t match an existing profile in CODIS or an identified suspect Provide information useful to the investigation improving the ability to identify potential suspects Will not directly identify a single suspect individual but will: assist in prioritizing suspect processing corroborate witness testimony assist in determining the relevance of evidence to a crime Dr. Joan Bienvenue 24

25 Forensic SNPs Individual Identification SNPs (IISNPs) Lineage Informative SNPs (LISNPs) Ancestry Informative SNPs (AISNPs) Phenotype Informative SNPs (PISNPs) SNPs that collectively give a high probability of an individual s ancestry being from one part of the world or being derived from two or more areas of the world. Podini Confidential Laboratory and Proprietary ZyGEM-MicroLab/Lockheed Forensic Sciences, Martin InformationGWU Dr. Joan Bienvenue 25

26 Ancestry Informative ISNPs - Duffy Null Allele Duffy blood group identifies phenotypes associated with two proteins that appear on the surface of red-blood cells Plays an important role in susceptibility to malaria infection The Fy(a-b-) phenotype (i.e., not expressing the receptor) represents an adaptation to living in malaria-endemic regions Predominant in African populations esp. those from West Africa. Podini Confidential Laboratory and Proprietary ZyGEM-MicroLab/Lockheed Forensic Sciences, Martin InformationGWU Dr. Joan Bienvenue 26

27 Forensic SNPs Individual Identification SNPs (IISNPs) Lineage Informative SNPs (LISNPs) Ancestry Informative SNPs (AISNPs) Phenotype Informative SNPs (PISNPs) SNPs that collectively give a high probability Of a phenotypic marker of a specific physical trait. Podini Confidential Laboratory and Proprietary ZyGEM-MicroLab/Lockheed Forensic Sciences, Martin InformationGWU Dr. Joan Bienvenue 27

28 Phenotype Informative SNPs Five genes (SLC24A, ASIP, MC1R, SLC45A2, OCA2) involved in pigment expression HERC 2 (part of OCA2) has SNPs strongly associated with blue eyes HERC 2 G. Tully, Forensic Science International: Genetics 1, 105 (2007 Podini Confidential Laboratory and Proprietary ZyGEM-MicroLab/Lockheed Forensic Sciences, Martin InformationGWU Dr. Joan Bienvenue 28

29 Next-Generation Sequencing Technologies Rapid sequencing of the entire genome of an individual Currently cost is $30-100K NIH searching for the technology for the $100 genome Dr. Joan Bienvenue 29

30 Next-Generation Sequencing Technologies Technologies are evolving rapidly for full genome seq (FGS) None capable of doing this in a costeffective way PacBio claims FGS 15 min for $100 by 2015 Dr. Joan Bienvenue 30

31 OUTLINE Future Directions for Biometrics DNA Microfluidics Where are we at? Where do we need to be? Beyond STRs: Next Generation Technological Advances SNPs NextGen sequencing New Technology, New Problems? Policy, ethics, legal, security concerns Dr. Joan Bienvenue 31

32 Policy, Security, and Ethical Questions - Microfluidics Taking DNA into the field, how do we maintain the quality and reliability of results? - What will the controls be? - How will instrument performance be monitored? - Validation of instrument, methods, chemistry? - Training and certification of users? - Audits, reviews, quality control measures? Automated data analysis, what happens when something goes wrong or the results are uninterpretable/need expert analysis? - Will data be shared with a laboratory for analysis? - Can data be transferred securely? Dr. Joan Bienvenue 32

33 Policy, Security, and Ethical Questions - Microfluidics What weight will the field results carry? - Will samples need re-analysis in the laboratory for court acceptance? - Can results be used to arrest, to hold, to convict? - State vs Federal vs Global, different standards, different access, different collection requirements? What policy has to change/be adapted to permit samples to be run in the field, in a booking station, at a checkpoint? - Can the data generated be uploaded to databases? - Can the data generated be used to search databases? - What samples are uploaded to databases? - Familial searching? Searching partial profiles? Dr. Joan Bienvenue 33

34 Policy, Security, and Ethical Questions SNPS, Sequencing, and Beyond NextGen racial profiling? - How and when do we use phenotypic genetic information? - Does ancestral/phenotypic testing violate civil rights? What other information do we get when we sequence and how do we prevent abuse? - Medical conditions? - What if genes are found that can be associated with a pre-disposition to violence? Murder? Sexual Assault? Dr. Joan Bienvenue 34

35 Acknowledgements Dr. Schuckers and the NSF for the invitation and opportunity to participate Grad students and post-docs in the Landers Laboratory at the University of Virginia Dr. Daniele Podini, George Washington University ZyGEM-MicroLab Scientists, Engineers and Management Lockheed Martin Technology Innovation Funding Dr. Joan Bienvenue 35