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Arthur J. Eisenberg, PhD Professor and Chair, Dept. of Forensic and Investigative Genetics Co-Director UNT Center for Human Identification Improved Methods for Extraction of DNA from Challenging Bone Samples

Improved Methods for Extraction of DNA from Challenging Bone Samples

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Presented at South Africa User Meeting, December 2012 by Arthur J. Eisenberg, PhD Professor and Chair, Dept. of Forensic and Investigative Genetics Co-Director UNT Center for Human Identification

Text of Improved Methods for Extraction of DNA from Challenging Bone Samples

  • 1. Improved Methods for Extraction ofDNA from Challenging Bone Samples Arthur J. Eisenberg, PhD Professor and Chair, Dept. of Forensic and Investigative Genetics Co-Director UNT Center for Human Identification

2. The UNT Center for Human Identification3 Divisions: Laboratory of ForensicAnthropology Laboratory for MolecularIdentification Forensic Services Unit / NamUsFocus: Missing and unidentifiedpersons Forensic casework for Texasagencies 3. The Problem Facing the United States Today in the United States there are over85,000 active missing persons cases. Almost half have a last known contact ofover a year ago Tens of thousands of individuals, bothchildren and adults, vanish each year undersuspicious circumstances. 4. The Problem Facing the United States Throughout the United States there may be40,000 or more skeletal remains stored atmedical examiners, coroners and lawenforcement agencies that cannot be identifiedby conventional means. Few crime laboratories in the United States areequipped to perform the DNA analysis of humanremains, especially when they are old orseverely degraded. 5. The Problem Facing the United States 50% or more of the unidentified decedents aremost likely homicide victims. If death was due to a homicide, and theremains disposed of without sample retention,there can be no accountability for theperpetrator. Advances in DNA technology could make itpossible for grieving families to obtainresolution and for those responsible (to) meetjustice. 6. University of North Texas Center for Human IdentificationA National Resource for the Identification ofMissing Persons and Unidentified Decedents 7. University of North Texas Center for Human IdentificationThe Center can provide: A family with information they need toobtain some measure of closure. Law enforcement with the critical first stepultimately leading to the identification ofthe perpetrator of the violent crime. 8. Free. Secure. Nationwide. www.namus.govThe National Missing and UnidentifiedPersons System offers law enforcementagencies, medical examiners, coroners,family members and victimadvocates a powerful tool forresolving missing andunidentified persons cases. 9. www.NamUs.gov 10. NamUs Automatic Searching 11. Forensic Services Available Through NamUs Forensic Odontologists on staff Fingerprint Examiner on staff DNA analyses through the UNT Center forHuman Identifications Laboratory for MolecularIdentification Coordination with local, state and federal DNAlaboratories across the country to affectcomparisons Forensic Anthropology through the UNT Centerfor Human Identifications Laboratory of ForensicAnthropology 12. Forensic Anthropology 13. Tools to Help Identify Missing Personsand Human Decedents Forensic Anthropological analysis of humandecedents to establish identity and to helpdetermine cause and manor of death Forensic Odontology and dental identificationthrough comparative analysis of the remains of adecedent and a known persons ante-mortemdental records Forensic Art to develop a facial post-mortemreconstruction of the decedent, and human agingtechniques and technologies 14. Initial Sampling of Bone at Laboratoryof Forensic Anthropology Cortical window: left tibia 15. Bone Sample Selection For DNA Analysis Typically chosen by theanthropologist Success of STR and mtDNAanalysis dependent on thequality of the remainsrecovered Prudent selection ofsample type will increasethe chance of success 16. Compact Bone Osteocytes are embedded and protected in compact microstructural spaces between concentric layers of bone material Characteristic of long bones Femur Tibiaarthursclipart.org 17. What Human Remain Samples Should be Submitted? Bones: most preferred to least preferred Long bones femur and tibia (green) Small bones humerus, radius, ribs, mandible, pelvis (light blue) Smaller bones vertebrae, ulna, metacarpals, fibula (dark blue) Clavicle, patella, metatarsals (yellow) Skull (pink)Profiles in DNA, March 2007, Suni Edson, AFDIL 18. Processing samples: Cutting Sample is moved to the Bone Cutting Lab Bone Cutting Lab is equipped with 3 custom designed hoods 19. Extraction of DNA from Human Remains 20. Extraction of DNA from Human Remains 21. Extraction of DNA from Human Remains 22. Extraction of DNA from Human Remains 23. DNA Profiles from Human Remains When human remains are found, they may bein a variety of conditions ranging from recentlydeceased to fully skeletonized The approach to obtaining DNA from remainscan differ, depending on the state of theremains 24. Remains with Significant Decomposition Most of the soft tissues will have lost theirintegrity Obtaining a DNA profile from liquefied tissuesis seldom successful Bone marrow can sometimes be betterpreserved and may provide sufficient DNA If unsuccessful, then recourse should be madeto skeletal structures 25. Remains That are Fully Skeletonized Only hardy structures such as bone, hair, nails andteeth will be available Skeletal structures, bone matrix and tooth pulp maycontain little or no amplifiable nuclear DNA but maybe rich in mtDNA It requires special procedures to release and purifyDNA encased within a hardened calcified matrix The yields of nuclear DNA are typically low and,often, elevated cycle number PCR (>28 cycles) isneeded to generate typing results 26. COmbined DNA Index System 27. CODIS INDICES OFFENDER Convicted Offenders Arrestee FORENSIC Forensic Crime Scene Samples MISSING PERSONS Unidentified Human Remains Missing Persons Direct Reference Samples (tooth brush, hair brush, baby teeth, etc.) Family Reference Samples 28. Missing Persons and UHR Datain CODIS as of November 1, 2012At NDIS: 1,081 missing persons profiles12,445 family reference profiles 6,411 remains profiles 5,978 pedigree trees: 3,216 with more than relative typedUNTCHIs portion:173 missing persons profiles (16% of NDIS)6,769 family reference profiles (54% of NDIS)3,098 remains profiles (48% of NDIS)4,484 pedigree trees (75% of NDIS): 2,339 with more than one relative typed (72% of NDIS) 29. Challenges Associated With the STRDNA Analysis of Human Remains Limited amounts of DNA available DNA samples are often highly degraded PCR inhibitors often co purify with DNA 30. STR Profiles From Degraded DNA Samples A multiplex STR amplification reaction cananalyse less than 200 pg of DNA, however, theDNA template must be intact where twoprimers bind as well as between the primers sothat full extension can occur The STR loci with larger sized amplicons in amultiplex amplification are the first to drop outof the DNA profile when amplifying extremelydegraded DNA samples 31. PCR Inhibition Another important challenge to amplifyingDNA samples from bones and crime scenes isthe fact that the PCR amplification process canbe affected by inhibition present in thesamples themselves Samples from crimes scenes and unidentifiedhuman remains may contain Taq DNApolymerase inhibitors 32. Examples of Inhibitors that canInterfere with PCR Amplification 33. PCR Inhibition Inhibitors can: Interfere with the cell lysis necessary for DNAextraction Inhibit Taq polymerase activity thus preventingenzymatic amplification of the target DNA Some inhibitors can co-extract and will remainwith the DNA through the entire isolationprocess 34. Strategies for Dealing with Degraded and Inhibited Samples Reduction in size of PCR amplicons Reducing the size of the PCR products will allowamplification from inhibited samples and maximize thechances of recovering information from samples wherethe DNA is severely fragmented. Optimization of reaction mix components tofacilitate the amplification of inhibited samples Improvements to buffer systems can provide the abilityto overcome inhibitors Optimization of extraction Ensuring effective removal of inhibitors Maximizing recovery of DNA per mg of bone sample 35. DNA Extract from Bone SampleAmplified with Identifiler 36. MiniSTR DevelopmentSmaller PCR products work better with low copy number or fragmented DNA templatesConventional miniSTRPCR primerprimer STR repeat region miniSTR Conventionalprimer PCR primer 37. AmpFlSTR MiniFiler Kit 38. Same Bone Sample DNA ExtractAmplified with MiniFiler System 39. AmpFSTR Identifiler Plus Improved PCR amplification over the current Identifilerkit providing increased sensitivity and robust results inthe presence of inhibitors Improved discrimination for casework samples Recover more complete DNA data as compared tocurrent version of Identifiler and other STR assaysystems Utilization of the same primer sequences as theoriginal Identifiler kit and the new Identifiler DirectKit eliminating concerns over discordant typing results 40. Bone Sample #1 DNA Extract Amplified with Identifiler 41. Bone Sample #1 DNA Extractnow Amplified with Identifiler Plus 42. NGM System Includes additional Non CODIS miniSTR loci150 bp200 bp D10S1048vWAD16 D2S1338Amel D8D21D18 D22S1045D19 TH01 FGA D2S441D3D1S1656 D12S391 43. Bone Sample Amplified with Identifiler 44. Bone Sample Amplified with the NGM STR System 45. Strategies for Dealing withOptimization of ExtractionEnsuring effective removal of inhibitorsMaximizing recovery of DNA per mg of bone samplePrepFiler BTA Lysis Buffer in conjunction with the AutoMate ExpressTM Benchtop Instrument 46. Automated Benchtop Forensic DNA Extraction System Core reagents are from the PrepFiler Forensic DNAExtraction Kit utilizing magnetic particle based nucleicacid purification technology PrepFiler BTA Lysis Buffer is a specialized DNAextraction buffer for extracting DNA from calcifiedtissues (bones, teeth), as well as certain adhesive andpaper-containing samples (some cigarettes, tapelifts) Optimized performance provides comparable orbetter DNA yields & purity than conventional phenol-chloroform-based methods 47. Automated Benchtop Forensic DNA Extraction System Fewer tube transfers and greater overall ease of useminimizes the opportunity for sample mix-ups orcontamination, while lessening the training burden ofnew analysts All required DNA purification reagents are in a sealeddisposable plastic cartridge 48. AutoMate ExpressTM Benchtop Instrument Processes 13 samples in about 30minutes Elution Vol: 50 L Two protocols (PFLB protocol and BTAprotocol) on a single script cardCartridge rackTip and tube rack 49. Cartridge Configuration Universal cartridge suitable for DNA extraction with PrepFiler BTA Lysis Buffer be be Tu Tu e npl iomutSaEl 1 2 3 4 5 6 7 8 9 10 11 12-10 Elution solutionLysis buffer MagneticWash solutionFor BTAParticlesprotocol Isopropanol(500ul) 50. PrepFiler Express BTABones/Teeth Sample ExtractionQuantifiler Duo ResultsQuantifiler Duo ResultsHumanHuman2.0120Male Total DNA yield (ng) 100Total DNA yield (ng)1.5 801.060 400.5 200.0050mg bone 10mg tooth 51. PrepFiler BTA Lysis Buffer andAutoMate ExpressTM Benchtop Instrument 52. PrepFiler BTA Lysis Buffer andAutoMate ExpressTM Benchtop Instrument 53. Overnight Demineralization and OrganicExtraction followed by QIAquick Spin Column 54. PrepFiler BTA Lysis Buffer andAutoMate ExpressTM Benchtop Instrument 55. PrepFiler BTA Lysis Buffer Bone ProtocolOptimizationIncubation Time + Amount of Bone Powder25.0Quantifiler TM Human Total Yield (ng)20.015.010.0 5.0 0.02h2h18h 18h2h 18h 50m g 50m g 50m g 50m g 200m g 200m g 56. PrepFiler BTA Lysis Buffer and AutoMate ExpressTM Benchtop InstrumentReproducibilty Quantification (ng/ul) Total DNA yield (ng)200mg Automate 1 4.22E-02 2.11200mg Automate 2 4.70E-02 2.35200mg Automate 3 4.69E-02 2.34200mg Automate 4 4.58E-02 2.29 57. PrepFiler BTA Lysis Buffer andAutoMate ExpressTM Benchtop InstrumentDifficult Older Bone Samples100 mg bone100 mg bone200 mg bone200 mg bonepowder powder powder powder (ng/ul) (ng/ul)H10B-H10C 106.61 Full profile 180.07 Full profileH12B-H12C1.29E-02Full profile4.54E-02Full profile 018-123.61E-03 Partial profile(12)1.57E-02 Partial profile(17) 029-121.13E-02 Partial profile(14)1.53E-02 Partial profile(23) 0020-12 2.09E-01Full profile1.81E-01Full profile003-14/201.32E-02 Partial profile(19)5.14E-02 Partial profile(23) 58. Sample H10B-10C (100mg) 59. Sample H12B-12C (100mg) 60. Arthur J. Eisenberg, PhD Professor and Chairman Dept of Forensic and Investigative GeneticsCo-Director UNT Center for Human Identification, Institute of Investigative Genetics University of North Texas Health Science CenterFort Worth, Texas USA 817 [email protected]