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Tools for Improving DNAExtraction EfficiencyHuman IdentificationLife Technologies
Efficient Solutions for Forensic DNA Analysis   PrepFiler® Forensic DNA Extraction Kit         −    Designed specifically ...
Hypothesis of DNA Binding to the PrepFiler®Magnetic Particles: Schematic Presentation                                     ...
PrepFiler® & PrepFiler® BTA DNA Extraction Chemistry:Designed for Wide Variety of Forensic Sample Types   Routine Sample T...
PrepFiler® Chemistry Platform Options   A single extraction chemistry available in several formats …        −   Provides ...
PrepFiler® Forensic DNA Extraction Kit         Options and Developments for Manual, Semi-Automated &         Automated Wor...
Alternative Chemistries Protocol Comparison                                                                               ...
PrepFiler® Kit: Comparison with Other Methods8/16/2012 | © Life Technologies™   8
PrepFiler® Manual Kit Updates New! PrepFiler® BTA Manual Forensic DNA Extraction Kit  Improves performance of DNA extract...
PrepFiler® BTA Kit: Protocol Highlights Optimized performance with bones, teeth, as well as other adhesive and paper-  co...
PrepFiler® BTA Manual Kit:Bone Sample: DNA Yield8/16/2012 | © Life Technologies™   11
PrepFiler® BTA Manual Kit:Bone Sample: Information Recovery8/16/2012 | © Life Technologies™   12
PrepFiler® BTA Manual Kit:Cigarette                                   Vendor 1        Vendor 28/16/2012 | © Life Technolog...
PrepFiler® BTA Manual Kit: Cigarette                                        Site A BTA                                    ...
Case Study 1: Missing Persons Case                                                                                        ...
Case Study 2: Identification of MissingPersons from Spanish Civil War                                   • Recovery of Hist...
Semi-Automated Extraction using the        AutoMate™ Express DNA Extraction System8/16/2012 | © Life Technologies™   17
The AutoMate Express™ Forensic DNA  Extraction System            AutoMate Express™ Instrument                           + ...
AutoMate Express™ DNA Extraction System  Tip and tube rack                              Cartridge                         ...
AutoMate Express™ DNA Extraction Workflow PrepFiler™ Lysis                                             PFLB or BTA Protoco...
The PrepFiler® LySep Column   Simple off-line Lysis & Substrate Removal                               Add Sample, Lysis   ...
Identifiler® Kit ProfilesRoutine Sample Types                                                                             ...
Identifiler® Kit ProfilesBone and Tooth Samples                                        270pg                              ...
PrepFiler® Express Extraction ProtocolProcessing of Sexual Assault Samples                                       E-cell   ...
Mock Sexual Assault SamplesQuantifiler® Duo Kit Results                                   E-Cell/Sperm Ratio, 20:18/16/201...
Identifiler® Kit Profiles                                         Sperm                                        Reference  ...
Correlation Study: PrepFiler® Express Kit                          120                                                    ...
Correlation Study: Total Allele Count                                                               AutoMate Express™     ...
PrepFiler® Express Kit UpdatesPlastic Consumable Cleanliness Initiatives All plastics, including PrepFiler® LySep columns...
PrepFiler® Automated Forensic DNA        Extraction Kit8/16/2012 | © Life Technologies™   30
The PrepFiler® Kit High Throughput AutomationSystem Multiple platform options now available to support the use  of the Pr...
Reproducibility and Concordance- Tecan                            DNA Yield     Experiment 1         Experiment 2         ...
PrepFiler® Kit Automated: Consistency of DNAQuality- Tecan          3000 RFU          3000 RFU          3000 RFU          ...
Cross-Contamination Study- TecanCheckerboard extraction of DNA from 6 blood donors (10 μL in replicates of 8) and 48 blank...
PrepFiler® DNA Extraction Verification – Hamilton                                                                         ...
PrepFiler® & PrepFiler® BTA Forensic DNA ExtractionChemistry: Summary New developments and improvements to support the us...
Thank YouFor Forensic or Paternity Use Only. Not for use in anyanimal or human therapeutic or diagnostic procedure.© 2012 ...
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Tools for Improving DNA Extraction Efficiency

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Transcript of "Tools for Improving DNA Extraction Efficiency"

  1. 1. Tools for Improving DNAExtraction EfficiencyHuman IdentificationLife Technologies
  2. 2. Efficient Solutions for Forensic DNA Analysis PrepFiler® Forensic DNA Extraction Kit − Designed specifically to maximize the yield and quality of DNA obtained from forensic samples − Provides superior DNA yield compared to organic methods and common commercial DNA extraction kits − Extracted DNA is optimized and validated for use with the Quantifiler® and AmpFℓSTR® kits to streamline integration − Protocols available for both manual and automated extraction to suit all laboratory throughput requirements8/16/2012 | © Life Technologies™ 2
  3. 3. Hypothesis of DNA Binding to the PrepFiler®Magnetic Particles: Schematic Presentation  PrepFiler® Magnetic Particles utilize a multi-component surface chemistry  DNA binds to the magnetic particles by formation of a complex between the polymer present on the surface of the particles, soluble polymer and DNA  The complex formation is accomplished in presence of high salt, detergent and isopropanol8/16/2012 | © Life Technologies™ 3
  4. 4. PrepFiler® & PrepFiler® BTA DNA Extraction Chemistry:Designed for Wide Variety of Forensic Sample Types Routine Sample Types Challenged Sample Types  Liquid samples  Bone and teeth − Blood, saliva and semen  Cigarette butt and chewing gum  Body fluid stains − Blood on cotton, rayon and nylon  Fingernail clippings, scrapings fabric, denim − Saliva on cotton fabric  Formalin fixed tissue − Semen on cotton fabric  Vaginal swabs w/ semen Touch Evidence-Type Samples  Pulled hair roots  Casework reference samples  Swabs from firearms/knives − Buccal swabs − Blood/Buccal on FTA® Card  “Wear area” swabs/cuttings  Steering wheel swab8/16/2012 | © Life Technologies™ 4
  5. 5. PrepFiler® Chemistry Platform Options  A single extraction chemistry available in several formats … − Provides flexibility in selecting a platform capable of required sample throughput − Supports more straightforward conversion to different platform, if throughput needs or applications should change  PrepFiler® Chemistry Platform Options − Manual − AutoMate Express™ DNA Extraction System − HID EVOlution™ System − Other Robotic Platforms (with scripts provided and validated/verified by HID Professional Services Team)8/16/2012 | © Life Technologies™ 5
  6. 6. PrepFiler® Forensic DNA Extraction Kit Options and Developments for Manual, Semi-Automated & Automated Workflows8/16/2012 | © Life Technologies™ 6
  7. 7. Alternative Chemistries Protocol Comparison Phenol PrepFiler®/ QIAamp DNA DNA IQ Chloroform PreFiler BTA ® Investigator (PCI) Purchase DTTPrelim/Prep Purchase DTT Purchase Proteinase K Purchase DTT Purchase Ethanol Purchase DTT Purchase Ethanol Purchase Proteinase K Custom Bone Buffer (BB) Purchase Isopropanol/Ethanol Custom Bone Buffer (BB) Make Bone Buffer (BB) Custom Incubation Buffer (IB) Custom Incubation Buffer (IB) Make Lysis Buffer (LB) T & H Kit T & H Kit Pre-soak in 0.5% SDS (E) 1 hr @ 56°C in BB (B, To) 40 mins-2 hr @ 56-70°C 30 min @ 56°C in LB (G, Ta) 18 hr @ 56°C in LB (B, To) 30 min @ 56°C in IB (C) 18 hr @ 56°C in BB (B, To) 40 mins @ 70°C 30 min @ 70°C in LB (E) 3 hr @ 56°C in LB (G)Lysis 1 hr @ 56°C in LB (C, Ta, E) 1.5 hr @ 56°C in LB (C, G, Ta, E) 1 hr @ 56°C in SB (B, To, G) 10 mins @ 56°C in SB (C, Ta, E) Add Binding SolutionCollection/Isolation Bind / Spin-Wash 3X Phenol:CHCl3:IAA Bind / Mag-Wash 3X YM30 Filter / Spin Elute DNA Elute DNA Elute DNA 8/16/2012 | © Life Technologies™ 7
  8. 8. PrepFiler® Kit: Comparison with Other Methods8/16/2012 | © Life Technologies™ 8
  9. 9. PrepFiler® Manual Kit Updates New! PrepFiler® BTA Manual Forensic DNA Extraction Kit  Improves performance of DNA extraction from calcified tissues, as well as certain adhesive and paper-containing samples − Bones and teeth − Cigarette butts Available − Envelope flaps, gum, tape Now8/16/2012 | © Life Technologies™ 9
  10. 10. PrepFiler® BTA Kit: Protocol Highlights Optimized performance with bones, teeth, as well as other adhesive and paper- containing samples; comparable or better yields & purity than conventional phenol- chloroform-based methods 50 to 200 mg of bone sample is optimal input material Involves fewer tube transfers and greater overall ease of use than conventional methods to minimize the opportunity for sample mix-ups or contamination 2 hour lysis time for bone samples compared to 18 to 48 hours by traditional and alternative commercial chemistry methods Simple, robust protocol facilitates training of users, and is more “tolerant” to range of experience levels Utilizes PrepFiler® chemistry for isolation of DNA and removal of PCR inhibitors8/16/2012 | © Life Technologies™ 10
  11. 11. PrepFiler® BTA Manual Kit:Bone Sample: DNA Yield8/16/2012 | © Life Technologies™ 11
  12. 12. PrepFiler® BTA Manual Kit:Bone Sample: Information Recovery8/16/2012 | © Life Technologies™ 12
  13. 13. PrepFiler® BTA Manual Kit:Cigarette Vendor 1 Vendor 28/16/2012 | © Life Technologies™ 13
  14. 14. PrepFiler® BTA Manual Kit: Cigarette Site A BTA Site A PCI Site B BTA Site B PCI8/16/2012 | © Life Technologies™ 14
  15. 15. Case Study 1: Missing Persons Case Figure 1: Bone sample extracted with QIAamp® Micro DNA kit and amplified with the Identifiler® kit. Neither the Identifiler® nor the Identifiler® Plus kits produced any STR results Figure 2: Bone extracted with the PrepFiler® BTA Figure 3: Bone extracted with the PrepFiler® BTA extraction kit and amplified with the Identifiler® kit. extraction kit and amplified with the Identifiler® Y scales between 200-400 RFU Plus kit. Y scales between 600-1000 RFU8/16/2012 | © Life Technologies™ 15
  16. 16. Case Study 2: Identification of MissingPersons from Spanish Civil War • Recovery of Historical Memory − Identification of victims from the Spanish Civil war − 24 bodies deposited in a well on March 19th, 1937 • PrepFiler® BTA Forensic DNA extraction chemistry combined with Next Generation DNA profiling kits to significantly improve the quantity and quality of results PrepFiler® BTA DNA Extraction and AmpFℓSTR® NGM™ kit8/16/2012 | © Life Technologies™ 16
  17. 17. Semi-Automated Extraction using the AutoMate™ Express DNA Extraction System8/16/2012 | © Life Technologies™ 17
  18. 18. The AutoMate Express™ Forensic DNA Extraction System AutoMate Express™ Instrument + PrepFiler® Express & PrepFiler® Express BTA Forensic DNA Extraction Kit  Developed specifically to enable maximum yield of high quality genomic DNA from forensic samples using the PrepFiler® Express Forensic DNA Extraction Kits − Processes up to 13 samples in ~30 minutes − PFLB and BTA protocols on a single script card8/16/2012 | © Life Technologies™ 18
  19. 19. AutoMate Express™ DNA Extraction System Tip and tube rack Cartridge rack8/16/2012 | © Life Technologies™ 19
  20. 20. AutoMate Express™ DNA Extraction Workflow PrepFiler™ Lysis PFLB or BTA Protocol Buffer (PFLB) Majority of samples including trace evidence samples, sexual assault samples – high extraction Lysate separation efficiency from substrate DNA for (including downstream differential lysate applications fractions if PrepFiler BTA™ necessary) Lysis Buffer Bone, tooth, sticky tape, DNA Binding cig. butt – for samples requiring delicate lysis Wash DNA Elution8/16/2012 | © Life Technologies™ 20
  21. 21. The PrepFiler® LySep Column Simple off-line Lysis & Substrate Removal Add Sample, Lysis Buffer & DTT* to Remove PrepFiler® PrepFiler® LySep LySep Column and Column discard Load on Instrument Incubate/ Shake High-speed centrifugation enables simple & efficient separation of lysate from substrate8/16/2012 | © Life Technologies™ 21
  22. 22. Identifiler® Kit ProfilesRoutine Sample Types 0.5 μL blood swab 0.3 μL blood on blue denim 0.3 μL blood w/inhibitors 1.0 μL semen on cotton E-cell swab All amplifications performed using 1 ng input DNA concentration8/16/2012 | © Life Technologies™ 22
  23. 23. Identifiler® Kit ProfilesBone and Tooth Samples 270pg 160pg 50 mg bone 190pg 1ng 1ng 10 mg tooth 1ng8/16/2012 | © Life Technologies™ 23
  24. 24. PrepFiler® Express Extraction ProtocolProcessing of Sexual Assault Samples E-cell PrepFiler® fraction Lysis buffer Standard PrepFiler® Express Differential PFLB Protocol extraction Sperm PrepFiler® fraction Lysis buffer The offline lysis protocol associated with the AutoMate Express™ DNA Extraction System allows for the same basic protocol, to be used across multiple sample types including differential extraction of sperm and e-cell fractions from sexual assault cases8/16/2012 | © Life Technologies™ 24
  25. 25. Mock Sexual Assault SamplesQuantifiler® Duo Kit Results E-Cell/Sperm Ratio, 20:18/16/2012 | © Life Technologies™ 25
  26. 26. Identifiler® Kit Profiles Sperm Reference Sperm Fraction E-Cell Fraction8/16/2012 | © Life Technologies™ 26
  27. 27. Correlation Study: PrepFiler® Express Kit 120 AutoMate Express™ System Method A 100 Method B 80 DNA Yield (ng) 60 40 20 0 Blood on cotton Blood on FTA E-cells on swab Blood on Extraction blank cloth paper (50 µL) blue denim (2 µL) (2 µL) (1 µL)8/16/2012 | © Life Technologies™ 27
  28. 28. Correlation Study: Total Allele Count AutoMate Express™ Method A Method B 100 90 % Alleles Recovered 80 70 60 50 40 Blood on cotton Blood on cotton Blood on FTA E-cells on swab Semen on cotton Blood on blue Bone cloth cloth paper (2 µL) (50 µL) cloth denim (50 mg) (0.3 µL) (2 µL) (1 µL) (1 µL)8/16/2012 | © Life Technologies™ 28
  29. 29. PrepFiler® Express Kit UpdatesPlastic Consumable Cleanliness Initiatives All plastics, including PrepFiler® LySep columns, are assembled and packaged in a clean room environment, with minimal human interaction Plastic components undergo “decontamination” by ethylene oxide for removal of any potential extraneous DNA These consumables are screened for the presence of any extraneous human DNA, using internal kit QC methods on every batch of kits produced, and by external testing conducted by an independent third party on a quarterly basis8/16/2012 | © Life Technologies™ 29
  30. 30. PrepFiler® Automated Forensic DNA Extraction Kit8/16/2012 | © Life Technologies™ 30
  31. 31. The PrepFiler® Kit High Throughput AutomationSystem Multiple platform options now available to support the use of the PrepFiler® Automated Forensic DNA Extraction Kit − HID EVOlution™ System (Tecan) − Forensics STARlet ABI Workstation (Hamilton) − Other Robotic Platforms (with scripts provided and validated/verified by HID Professional Services Team) Suitable for high throughput operations where releasing analyst time for more operator-dependent processes is critical Key parameters: reproducibility, consistency and minimal risk of contamination Packaging improvements for the manual PrepFiler® Kit have also been applied to the automated kit8/16/2012 | © Life Technologies™ 31
  32. 32. Reproducibility and Concordance- Tecan DNA Yield Experiment 1 Experiment 2 Experiment 3 Manual Extraction IPC CT Experiment 1 Experiment 2 Experiment 3 Manual Extraction 6 40 DNA Yield (ng/mL) 30 4 IPC CT 20 2 10 0 0 5 μL Blood 50 μL Saliva 1 μL Semen 5 μL Blood Stained 5 μL Blood Extraction Stained Cotton on Swab Stained Cotton Cotton + Inhibitors Stained Denim Blank Sample TypeSamples were run in replicates of 8 and 3 for automated and manual operations, respectively8/16/2012 | © Life Technologies™ 32
  33. 33. PrepFiler® Kit Automated: Consistency of DNAQuality- Tecan 3000 RFU 3000 RFU 3000 RFU 3000 RFU 3000 RFU 3000 RFU 3000 RFU 3000 RFU Automated extraction of DNA from 8 challenging samples - 2 µL of blood stain on cotton in presence of inhibitors8/16/2012 | © Life Technologies™ 33
  34. 34. Cross-Contamination Study- TecanCheckerboard extraction of DNA from 6 blood donors (10 μL in replicates of 8) and 48 blanksamples (concentration shown in ng/µL) 1 2 3 4 5 6 7 8 9 10 11 12 A 10.82 0.00 9.77 0.00 11.06 0.00 16.63 0.00 11.63 0.00 14.76 0.00 B 0.0004 19.93 0.00 11.64 0.00 9.25 0.00 10.37 0.00 12.52 0.00 11.12 C 14.99 0.00 13.34 0.00 20.96 0.00 11.07 0.00 8.86 0.00 7.26 0.00 D 0.00 10.80 0.00 16.03 0.00 13.67 0.00 20.76 0.00 10.49 0.00 8.98 E 15.38 0.0005 8.74 0.00 9.94 0.0004 14.23 0.00 12.80 0.00 14.84 0.0006 F 0.00 20.61 0.00 11.83 0.00 8.57 0.00 9.49 0.00 15.45 0.00 9.74 G 14.46 0.0005 11.72 0.00 19.65 0.0009 10.60 0.00 8.41 0.00 5.64 0.00 H 0.00 7.22 0.00 15.71 0.00 11.92 0.00 19.72 0.00 11.28 0.00 10.67 None of the blank wells exhibited an STR profile when amplified with the MiniFiler™ kit8/16/2012 | © Life Technologies™ 34
  35. 35. PrepFiler® DNA Extraction Verification – Hamilton Blood on Cotton Saliva on Cotton Diluted Blood on Swab Diluted Saliva on Swab Indirect Dried Saliva Mock sample study – NGM SElect™ Kit profiles8/16/2012 | © Life Technologies™ 35
  36. 36. PrepFiler® & PrepFiler® BTA Forensic DNA ExtractionChemistry: Summary New developments and improvements to support the use of the PrepFiler® chemistry for all types of forensic DNA extraction − PrepFiler® and PrepFiler® BTA kits available for manual and AutoMate Express™ workflows to provide maximum flexibility for routine and challenging sample types − Introduction of new wash buffer and kit packaging changes to improve performance, usability, and ordering experience − PrepFiler® kit available for fully automated workflows on different platforms such as Tecan and Hamilton to support high throughput of routine sample types − HPS available to facilitate validation & implementation of various HT platforms − New initiatives introduced to improve cleanliness of AutoMate Express™ plastics8/16/2012 | © Life Technologies™ 36
  37. 37. Thank YouFor Forensic or Paternity Use Only. Not for use in anyanimal or human therapeutic or diagnostic procedure.© 2012 Life Technologies Corporation. All rightsreserved. The trademarks mentioned herein are theproperty of Life Technologies Corporation or theirrespective owners.
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