HID University Seminar Series Intro & Evolution of Forensic DNA
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HID University Seminar Series Intro & Evolution of Forensic DNA



HID University Seminar Series Intro & Evolution of Forensic DNA

HID University Seminar Series Intro & Evolution of Forensic DNA



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HID University Seminar Series Intro & Evolution of Forensic DNA Presentation Transcript

  • 1. Future Trends in Forensic DNATechnology Seminar SeriesIntroductionHuman IdentificationLife Technologies
  • 2. 1 Company / 8 Trusted Brands / 600,000+Citations GROUNDED in Science INSPIRED by Scientists DRIVEN by Innovation8/16/2012 2
  • 3. Quick Facts8/16/2012 3
  • 4. Media Recognition Red Dot 2012 Award for Ranked Among Excellence In Product Design “The 50 Most Innovative Companies in 2012” Ranked #9 in 2012 Austin Plant Awarded 318 Chip Awarded for Best Plant in 2012 Ranked #15 in 2012 Design & Engineering In Embedded Technologies8/16/2012 4
  • 5. Ion Semiconductor SequencingRapid, Benchtop Sequencing for All8/16/2012 5
  • 6. This is Why We’re Here Freedom for the Innocent Solutions to Epidemics Life Changing Medicine “When you are in prison, and you “Whole-genome sequencing has are told that you’ve got to be “the new super toxic E. Coli enabled doctors to provide the there for the rest of your life - for strain was decoded in just two Beery twins with a simple, something you didn’t do – hours – a marvel to scientists” highly effective treatment for a DNA testing said otherwise” June 2011 rare condition” June 2011 Herman Atkins, Exonerated February 20008/16/2012 6
  • 7. This is Why We’re Here Reported rapes have fallen to the lowest level in 20 years as DNA evidence helps send more rapists to prison USA Today, October 7, 20098/16/2012 7
  • 8. Combating Human TraffickingA Major Global Problem An estimated 600,000 to 820,000 men, women, and children are trafficked across international borders each year, approximately 80 percent are women and girls and up to 50 percent are minors – US State Department 12.3 million adults and children in forced labor, bonded labor, and commercial sexual servitude at any given time…. estimated 56 percent of all forced labor victims are women and girls – International Labor Organization (United Nations) The human trafficking phenomenon affects virtually every country, including the United States – Hillary Clinton, US Secretary of State8/16/2012 8
  • 9. Use of DNA Testing to Prevent Human Trafficking In January 2011, more than 70 people including 25 children from Haiti arrived at an airport in Bolivia from Lima. Visa problems stopped them on their way to Brazil or Argentina Bolivian Police suspicions opened an investigation. The Bolivia Forensic Research Institute, a DNAProkids laboratory, performed DNA testing using Bode swabs and the Identifiler® Direct Route Taken to kit Bolivia from Haiti All the children were being trafficked: 13 were returned to their families in Haiti who were searching for them 8/16/2012 9
  • 10. 8/16/2012 10
  • 11. Customer Product CommunicationSystemA web-based system that ensures the timely delivery, and real-time reporting, of important technical product information toappointed contacts at each North America customer site.Launched in North America October 2011 Communication about critical product quality issues affecting specific HID customers: product retirement, product discontinuation, new software version or patch release, user bulletin, and non-critical technical updates Not a marketing channel! To sign up, go to: http://marketing.appliedbiosystems.com/mk/get/ HID_Product_Notification_Sign_Up8/16/2012 11
  • 12. Minor Manufacturing Changes to theAmpFSTR® PCR Amplification KitsNew Vendor Qualification Program
  • 13. Minor Modifications to Several AmpFSTR® Kits Manufacturing of the AmpliTaq Gold® enzyme and 10X PCR Buffer II components transitioning from Roche to Life Technologies − Both components in the following kits: Profiler®, Profiler Plus®, Profiler Plus® ID, SGM Plus®, COfiler®, Identifiler®, Yfiler® & MiniFiler™ Kits − The buffer component only in the Identifiler® Direct Kit (this kit already contains the AmpliTaq Gold® enzyme manufactured by Life Technologies) − Manufacturing of both components by Life Technologies will be conducted according to the same specifications used previously by Roche. − All other kits already contain both the buffer and enzyme components manufactured by Life Technologies8/16/2012 13
  • 14. Minor Modifications to Several AmpFSTR® Kits Our internal studies show the kits containing the components manufactured by Roche and Life Technologies to be interchangeable − The buffer and enzyme components manufactured by Life Technologies have already been tested rigorously during developmental validation of the Identifiler® Plus, NGM™ and NGM SElect™ Kits User Guides and/or Product Inserts for each of the affected kits will be updated to include our internal developmental validation studies demonstrating no substantive difference in performance compared to previous versions A full revalidation should not be necessary; however, laboratories should determine their own implementation requirements8/16/2012 14
  • 15. Minor Modifications to Several AmpFSTR® Kits Transition Process − Expected to commence in September 2012 − New and current versions will be available simultaneously for approximately 3 months to allow customers to transition smoothly − All kits will maintain the same kit names and part numbers > The first batch of kits containing buffer and enzyme components manufactured by Life Technologies can be distinguished from earlier versions by the letters “NF” after the lot number Why are we doing it? − To exercise greater control over the quality of key raw materials − To prevent formulation or QC issues which affect kit performance − To better maintain reagent supply8/16/2012 15
  • 16. Forensic News  Spring 2012 Edition − www.appliedbiosystems.com/forensicnews − The latest information on human identification applications, product updates, unique laboratory applications and new technologies  Includes articles on: − Tools for Improved DNA Extraction Efficiency − Expanding the Capabilities of Direct Amplification − Sample Collection Solutions from Life Technologies − Coming Soon...The Worlds Most Powerful STR Kit − Identifiler® Plus Kit Increases Success Rate of DNA Typing from Challenging Casework Samples by as much as 30% − MiniFiler™ Applications to Human Identification and Forensic Casework Analysis − Case Study: Y Testing at Court—AmpFSTR® Yfiler® Kit Drives the Success of Y Chromosomal Analysis in Forensic Genetics − Considerations for the Evaluation of Plus Stutter for AmpFSTR® PCR Amplification Kits in Human Identification Laboratories8/16/2012 16
  • 17. New Life Tech App – Technical Document App “Coming Soon” to an App Store near you Sample - Search for COA ACTIVATE AIM RESULTS (Touch) (Autoscan) (COA info)8/16/2012 17
  • 18. Thank You!! © 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.8/16/2012 18
  • 19. Evolution of Forensic DNAHuman Identification, Life Technologies
  • 20. A look back… 10 years ago  9/11 WTC Attacks  CSI only in 2nd Season, but surging in popularity8/16/2012 20
  • 21. A look back… 10 years ago  Fragmented, Inefficient Forensic DNA Workflow − Manual Processes − Quantiblot − GeneScan®/Genotyper® Software  Minimal Use of Y-STRs  Limited options for analyzing heavily degraded samples  Global DNA Databases in their infancy (except for the UK)8/16/2012 21
  • 22. Common beliefs I encountered… “Automation cannot work for forensic DNA casework.” “Manual Organic Extraction is the only trusted method for casework.” “Automated data analysis with advanced software (e.g. expert systems) is impossible.” “Analysis of lesser felonies i.e. property crimes is not practical.”8/16/2012 22
  • 23. Increased Discrimination and Sensitivity/Robustness ~1 in 1018 (quintillion) Bone sample amplified using the Identifiler® KitDiscrimination Power Bone sample amplified using the NGM™ KitMean Peak Height (RFU)8/16/2012 23
  • 24. Today Increased Integration and Efficiency……. Increased Efficiency Success with Simplified Higher StreamlinedEase of Use Difficult Samples Interpretation Throughput Workflow8/16/2012 24
  • 25. Streamlined Workflows for Single Source Samples Identifiler® Direct NGM SElect™ Kit Express Kit Prep-n-Go™ Buffer8/16/2012 25
  • 26. Improved Efficiency for Casework Samples Sample Quantitation/ Amplification Detection Analysis Preparation Normalization Dependent on sample type and method Time to Result 1-48 2 3.5 0.75 2-3 (hrs/per 6-16 samples) Total Time to Result = ~10-58 hrs8/16/2012 26
  • 27. Improved Efficiency for Casework Samples Sample Quantitation/ Amplification Detection Analysis Preparation Normalization Time to Result 1-48 1-3 2 3.5 1.5 0.75 0.55 2-3 1-2 (hrs/per 6-16samples) (hrs/per 13 samples) Total Time to Result == ~6-9 hrs Total Time to Result ~10-58 hrs8/16/2012 27
  • 28. Evolution of STR Technology 1996 AmpFSTR® PCR Amplification Kits 2012 Triplexes Larger Multiplexes Enhanced Capability Optimized for Application Blue Profiler® SEfiler™ Plus NGM™ Green I Profiler Plus® Yfiler® NGM SElect™ Expanded Profiler Plus® ID MiniFiler® NGM SElect™ Express “Global” COfiler® Identifiler® Plus Multiplexes SGM Plus® Identifiler® Direct Identifiler® SEfiler™ A comprehensive product portfolio, developed, validated and manufactured according to stringent performance standards specifically for use on forensic samples8/16/2012 28
  • 29. UNITED STATES LEGISLATIVE WAVE ALL CONVICTED OFFENDER LEGISLATION 1999 – 5 states 2003 – 27 states 2008 – 42 states 2011 – 50 states ARRESTEE LEGISLATION 1999 – 1 state 2006 – 7 states 2011 – 26 states8/16/2012 29
  • 30. THEADOPTS DATABASING WORLD 44 COUNTRIES, OVER 35 MILLION OFFENDER SAMPLES Fully implemented programs Legislation passed, programs not yet implemented Australia Estonia Netherlands South Korea Austria Finland New Zealand Spain Barbados France Macedonia Sweden Belarus Germany Norway Switzerland Belgium Hong Kong Panama Taiwan Brazil Hungary Poland United Arab Emirates Canada Iceland Portugal United Kingdom Chile Israel Qatar United States China Japan Russia Croatia Jordan Slovenia Cyprus Kuwait Slovakia Denmark8/16/2012 Latvia Singapore 30
  • 31. Progress Towards Cross-Border Data Exchange Prüm Treaty DNA Implementation Status 11-11-118/16/2012 31
  • 32. Global Databases: Locus Overview CODIS CODIS Locus Europe CODIS Locus Europe CODIS Proposed Proposed TPOX X* X D2S1338 X X X* CSF1PO X* X* D19S433 X X X* D5S818 X* X* D12S391 X* X* D7S820 X* X* D1S1656 X* X* D13S317 X* X* D2S441 X* X* FGA X* X* X* D10S1248 X* X* vWA X* X* X* D22S1045 X* X D3S1358 X* X* X* SE33 X X D8S1179 X* X* X* DYS391 X* D18S51 X* X* X* * Required loci D21S11 X* X* X* Loci in bold contained in either the Identifiler® or TH01 X* X* X* NGM SElect™ Kits D16S539 X X* X*8/16/2012 32
  • 33. The World Is Positioning For Explosive Growthof DNA Databases Dr. Reddy, Laboratory Director CDFD, India8/16/2012 33
  • 34. The World Is Positioning For Explosive Growthof DNA Databases  INDIA  COLOMBIA  INDONESIA  ARGENTINA  BRAZIL (LEG. PASSED)  KENYA  PAKISTAN  PERU  NIGERIA  SAUDI ARABIA  RUSSIA (LEG. PASSED)  MALAYSIA (LEG. PASSED)  MEXICO  VIETNAM  TURKEY  THAILAND  ITALY (LEG. PASSED)  SOUTH AFRICA “Currently there are about 35 trained analysts for DNA profiling [in all of India]. The country will need about 1,100 analysts [over the next 10 years].”8/16/2012 34
  • 35. Increasing Casework Backlogs U.S. Casework Backlogs  Backlogs increasing with Increased submissions  Samples increasing in complexity  Shortage of trained analysts  Validation and implementation barriers Source: NIJ Special Report “Making Sense out of DNA Backlogs, 2010 – Myths vs. Reality” www.ojp.usdoj.gov/nij To help address these and other challenges, we want to provide more than just core technologies8/16/2012 35
  • 36. HID Service Solutions HID Professional Services Program (HPS) − Delivery of internal validation programs and/or performance checks on any Applied Biosystems HID product − Robotic Validation/Implementation Support “HID University” Training Programs − Comprehensive suite of training courses designed to provide continuing education to forensic DNA analysts LIFE Center for Forensic Excellence at UNTHSC − Helps developing nations to rapidly establish world-class database laboratories through a unique certificate training program Advisory Complete Laboratory Analyst Training On-site Support and Consulting Integration and Validation Programs Training8/16/2012 36
  • 37. Looking ahead…Potential PGM™ Human Identification Applications Mitochondrial Phenotypic SNPs for SNP Genotyping DNA Sequencing Investigative Leads Paternity, Mixtures, Missing Missing Persons, DVI Persons, DVI Eye Color Facial Reconstruction Hair Color8/16/2012 37
  • 38. 21st Century Crime Fighting...DNA profiles of all felony arrestees loaded to aninvestigative database8/16/2012 38
  • 39. 21st Century Crime Fighting...DNA used to investigate all typesof evidence8/16/2012 39
  • 40. 21st Century Crime Fighting... Data shared globally across national databases8/16/2012 40
  • 41. 21st Century Crime Fighting...Rapid turnaround time of DNA evidence Data shared globally across national databases8/16/2012 41
  • 42. Our Role Enable Forensic Labs to Do More with Less • Increase efficiency with next-gen products and workflow integration (more info, less time) • Improve performance on difficult and compromised samples (more info, less sample) • Be a true systems integration partner (leveraging HID Professional Services) • Enable use of latest technologies for forensic and biometric applications8/16/2012 42
  • 43. Thank You © 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners For Forensic & Paternity Use Only8/16/2012 43
  • 44. The Evolution of Direct Amplification:From Sample to Result in 2 Hours*Human Identification, Life Technologies * Typical workflow for 24 samples
  • 45. Traditional Single Source Sample Workflow Sample Quantitation/ Collection Amplification Detection Analysis Preparation NormalizationTime to Result ~1-3.5 ~0-2 ~4 ~1.5 ~1(hrs/per 24 samples) Total Time to Result = ~7-12 hrs8/16/2012 45
  • 46. Development of Direct Amplification on Treated Paper Substrates8/16/2012 46
  • 47. Evaluation of Direct Amp Capability of theIdentifiler® Kit Control DNA 00725 μL Reaction Volume 1.2 mm Blood on FTA® Disc No full profiles under direct amp conditions8/16/2012 47
  • 48. Effect of Reducing Disc Size on Identifiler® Kit DirectAmp Performance Control DNA 007 (1 ng) But: − No automated 0.5 mm punch head options 0.5 mm blood − 0.5 mm disc on FTA® disc size too small for buccal samples − 0.5 mm discs very hard to0.75 mm blood handle on FTA® disc 1.2 mm blood on FTA® disc 8/16/2012 48
  • 49. Identifiler® Kit Direct Amp Performance Evaluation Possible explanations for poor performance − Insufficient DNA? 1 μL blood ~ 50 ng DNA Height = 1mm 1.2 mm disc ~ 56.52 ng DNA 0.75 mm disc ~ 22.08 ng DNA 0.5 mm disc ~ 9.81 ng DNA − Excess PCR inhibitors? 0.5 mm disc ~ 9.81 ng DNA Sufficient DNA still available Inhibitor concentration reduced by >5-fold8/16/2012 49
  • 50. Optimization of Reaction Buffer for 1.2 mm Discs Use of Design of Experiments (DOE) Approach Response Component 2 Component 18/16/2012 50
  • 51. Optimization of Identifiler® Direct Kit Master Mix Buccal sample on 1.2 mm FTA® disc before DOE8/16/2012 51
  • 52. Optimization of Identifiler® Direct Kit Master Mix Buccal sample on 1.2 mm FTA® disc after DOE8/16/2012 52
  • 53. Effect of Punch Position on Sample Peak Heights − 3 x liquid blood samples − 80 μL of blood onto FTA® Classic (passive diffusion) − Sample 1.2 mm discs from the center to the edge of the blood stain − Perform replicates for each position 1 2 34 58/16/2012 53
  • 54. Direct Amplification from Treated Paper Blood on FTA® samples Identifiler® Kit Example 1 FTA® Classic Card Identifiler® Direct Kit Identifiler® Kit Example 2 Identifiler® Direct EasiCollect® System Kit8/16/2012 54
  • 55. Identifiler® Direct Kit Validation: External Test Site Results PCR Success Rate Interpretation Success Rate Number of Sample Type Range of Mean Success Range of Mean Success Samples Tested Success rates Rate Success rates Rate Blood VTS Study 99.4 % 99.4 % 95.7 – 98.8% 97.3 % 414 Buccal 91.8 – 99.4% 97.1 % 84.2 – 95.5% 90.9 % 653 Blood CTS Study 100% 100 % 98.8 – 100% 99.8 % 437 Buccal 98.7 – 100% 99.0 % 91.7 – 100% 94.7 % 703  1st Pass Success Rate Definition − All profile peaks higher than specified threshold − Off scale peaks produce no artifacts which interfere with profile interpretation > OL-labelled pull-up peaks <20% of highest peak of the marker > No split, double called peaks > Stutter peaks < 20% of marker or < marker stutter cut off, whichever is higher8/16/2012 55
  • 56. Expansion of the Direct Amplification Workflow to Non-FTA Substrates Untreated Paper8/16/2012 56
  • 57. Expansion of the Direct Amplification Workflow toNon-FTA® Substrates Identifiler® Direct Kit development and optimization performed on FTA® substrates only Laboratories using untreated paper or swab substrates and/or alternative marker sets looking to recognise time and cost savings of the Direct Amplification workflow Goal: Enable direct amplification of non-FTA® buccal samples Minimal additional workflow steps Prep-n-Go™ High quality, well balanced profiles Buffer No introduction of artifacts8/16/2012 57
  • 58. Direct Amp Workflow: Untreated Paper Substrates Collect Samples on Untreated Paper Untreated paper One New Step Add 2 µL Prep-n-Go™ Buffer protocol development performed on Bode Punch 1.2 mm disc Buccal Collector™ Add PCR reagents Amplify Electrophorese8/16/2012 58
  • 59. Identifiler® Direct Kit Amplification of Bode Buccal DNA Collector™ Samples lysed with Prep-n-Go™ Buffer 2 different individuals amplified for 26 PCR cycles Well-balanced profiles within each dye color8/16/2012 59
  • 60. Performance Study Results Summary Number of PCR PCR Success Rate Interpretation Success Rate CE Test Site Samples Cycle Platform Number of First Pass Number of First Pass Tested Number Full Profiles Success Rate Full Profiles Success Rate 1 82* 27 3130xl 81/82 98.8% 78/82 95.1% 2 80 26 3130xl 78/80 97.5% 78/80 97.5% 3 84* 26 3130xl 83/84 98.8% 79/84 94.0% 4 84* 26 3130xl 80/81 98.8% 76/81 93.8% 5 84* 26 3730 83/84 98.8% 83/84 98.8% 6 84 26 3500 82/84 97.6% 74/84 88.1% Life Technologies 84 26 3130xl 82/84 97.6% 79/84 94.0% Total 582 569/582 97.8% 547/582 94.0% * Used real offender samples8/16/2012 60
  • 61. Performance Study: Bode Buccal DNA Collector™ Samples lysed in Prep-n-Go™ Buffer Peak Heights Intracolor Balance All amplifications performed using the Identifiler® Direct Kit8/16/2012 61
  • 62. Lysis Buffer Performance Comparison Identifiler® Direct Kit w/Other Buffer Identifiler® Direct Kit w/ Prep-n-Go™ Buffer1200 1200 No allelic drop-out using Prep-n-Go™ Buffer8/16/2012 62
  • 63. Expansion of the Direct Amplification Workflow to Non-FTA Substrates Buccal Swabs8/16/2012 63
  • 64. Buccal Swabs Pros − Easy to use Foam Swab − Inexpensive − Multiple types available Omni Swab Cotton Swab Flocked Swab T-Swab8/16/2012 64
  • 65. Buccal Swabs Cons − High performance variability among swab types − Donor variation − Sample collection techniques and storage conditions Donor Mouth Conditions − Limited opportunities for Collection Technique automation increasing labor requirements and the risk of contamination Drying & Transport Long-Term Storage Conditions Swab Age8/16/2012 65
  • 66. Swab Structure Comparison Cotton Swab Flocked Swab 2 km microfiber 6 m microfiber Sample stays Sample is released entrapped in the quickly and in fiber wand higher amounts8/16/2012 66
  • 67. 4N6FLOQSwabs™ by Copan  Excellent recovery of DNA  Maximum efficiency in collection capacity  Available with different anatomical and ergonomic designs  Certified free of Human DNA, Dnase and RNase  ETO-treated (Ethylene Oxide) FLOQSwabs™ consist of shortNylon® fibers that are arranged Distributed by in a perpendicular fashion8/16/2012 67
  • 68. Direct Amp Workflow: Identifiler® Direct Kit Collect Samples on Buccal Swab Incubate in oven Lyse swab in 400 μL Prep-n-Go™ Buffer adaptor at 99°C for 20 minutes New steps Add PCR Reagents Transfer Lysate Amplify Electrophorese8/16/2012 68
  • 69. Performance of Aged Swabs: Identifiler® Direct Kit& Prep-n-Go™ Buffer 4N6FLOQSwabs™ Omni Swabs Puritan Swabs Mean time between collection and analysis: 90 days; Amplification: 26 cycles8/16/2012 69
  • 70. Expansion of the Direct Amplification Workflow to Other STR Marker Sets8/16/2012 70
  • 71. Direct Amplification with the NGM™ & NGM SElect™ Kits NGM™ Kit NGM SElect™ Kit Amplification of buccal samples on Bode Buccal Collector™ treated with Prep-n-Go™ Buffer8/16/2012 71
  • 72. Direct Amplification for the Expanded ESSL AmpFSTR® NGM SElect™ Express Kit Utilizes the same primer sequences as the NGM™ and NGM SElect™ Kits Includes a new master mix optimised specifically to support direct amplification of swab and treated/untreated paper substrates Delivers rapid cycling times through the introduction of a new fast-capable enzyme (< 1 hr) May be amplified using the Veriti® 96-Well or 9700 (silver or gold-plated silver block) thermal cyclers − Veriti® 96-Well Thermal Cycler (standard) now supported for use with all existing AmpFSTR® kits8/16/2012 72
  • 73. Direct Amp Workflow: NGM SElect™ Express Kit Collect Samples on Buccal Swab Heated lysis optional Lyse swab in 400 μL Prep-n-Go™ Buffer depending upon swab age/type New steps Add PCR Reagents Transfer Lysate Amplify Electrophorese8/16/2012 73
  • 74. AmpFSTR® NGM SElect™ Express KitExample Profiles: Treated Paper Buccal on Copan NUCLEIC-CARD™ Blood on FTA® Classic Card8/16/2012 74
  • 75. AmpFSTR® NGM SElect™ Express KitExample Profiles: Untreated Paper Buccal on Bode Buccal Collector™ Blood on 903 Paper8/16/2012 75
  • 76. AmpFSTR® NGM SElect™ Express KitExample Profiles: Swabs Buccal on Whatman Omniswab Buccal on Copan Flocked Swab8/16/2012 76
  • 77. Test Site Results Profile Assessment Off Scale Profiles Partial Profile First Pass Success Rate Test Sample Cycle CE No Profile N Site Type # Platform 50 RFU 175 RFU 450 RFU 50 175 450 RFU RFU RFU # % # % # % 3 Copan 25 3500xL 60 1 1 1 1 1 58 97 58 97 58 97Reproducibility Copan 4 26 3100 60 0 4 N/A 1 37 58 97 58 97 NA NA (2 weeks) Copan 5 25 3130xl 60 0 0 N/A 0 8 60 100 60 100 NA NA (Fresh) 3 Omniswab 27 3500xL 40 1 2 3 1 0 37 93 36 90 35 88Performance Prionics 4 26 3100 40 0 2 N/A 1 31 39 98 39 98 NA NA (2 months) 5 Omniswab 26 3130xl 42 0 1 N/A 0 7 42 100 42 100 NA NA 8/16/2012 77
  • 78. 8/16/2012 78
  • 79. Factors Influencing Direct Amplification Results Choice of kit Choice of sample type/substrate Sample Transfer Efficiency Punch Size and Position Cycle Number Data Analysis Parameters8/16/2012 79
  • 80. Maximising Direct Amplification Results Swab Substrate Handling − Ensure swabs are fully dried before storing − Ensure swabs are stored correctly to prevent excessive degradation over time Swab Lysate Handling − If using a 96-well deep well plate for lysis, remove lysate from swab heads when aliquotting for storage and discard the plate containing the swab heads to reduce contamination risk − Avoid taking cell debris or precipitation from the bottom of the lysate tube when transferring swab lysate to amplification or storage plates/tubes − Follow the kit-specific instructions for lysis and amplification > Heated lysis may improve performance dependent upon direct amplification kit, swab age and type8/16/2012 80
  • 81. Maximising Direct Amplification Results Thermal Cycling Platform − Life Technologies kits optimised for use on the 9700 with silver or gold- plated silver block and the Veriti® 96-Well thermal cycler only > Not supported for use on the 9700 with Aluminium block or > Not supported for use on the Veriti 96-well Fast thermal cycler Choice of Cycle Number − Choose a cycle number that prevents allele drop-out and minimizes off- scale alleles − Use of elevated cycle numbers may cause presence of artifacts Optimisation of Software Analysis Settings − Use of settings appropriate to single source samples will reduce editing requirements and facilitate expert system analysis8/16/2012 81
  • 82. Optimising Data Analysis Parameters for Single-Source Samples  Use of a global cut-off filter can reduce significantly the amount of editing required for single-source sample data  Peak Quality settings may also be adjusted to better reflect the characteristics of single-source samples8/16/2012 82
  • 83. Optimized Parameters = More Efficient Analysis Unoptimized Optimized 8/16/2012 83
  • 84. Maximized Efficiency for Single SourceSample Processing Quantitation/ Collection Extraction Amplification Detection Analysis NormalizationTime to Result ~1-3.5 ~0-2 ~4 ~1.5 ~1(hrs/per 24 samples) Total Time to Result = ~7-12 hrs8/16/2012 84
  • 85. Maximized Efficiency for Single SourceSample Processing Collection Amplification Detection AnalysisTime to Result ~1-3.5 0 ~0-2 0 0.75 ~4 0.55 ~1.5 0.1 ~1(hrs/per 24 samples) Total Time toto Result = ~2 hrs Total Time Result = ~7-12 hrs8/16/2012 85
  • 86. 8/16/2012 86 *Kits currently in development
  • 87. Thank You © 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners For Forensic or Paternity Use Only.8/16/2012 87