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Improved Methods for Extraction of DNA from Challenging Bone Samples

Improved Methods for Extraction of DNA from Challenging Bone Samples



Presented at South Africa User Meeting, December 2012 by Arthur J. Eisenberg, PhD ...

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



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    Improved Methods for Extraction of DNA from Challenging Bone Samples Improved Methods for Extraction of DNA from Challenging Bone Samples Presentation Transcript

    • 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
    • The UNT Center for Human Identification3 Divisions: • Laboratory of Forensic Anthropology • Laboratory for Molecular Identification • Forensic Services Unit / NamUsFocus: • Missing and unidentified persons • Forensic casework for Texas agencies
    • The Problem Facing the United States• Today in the United States there are over 85,000 active missing persons cases.• Almost half have a last known contact of over a year ago• Tens of thousands of individuals, both children and adults, vanish each year under suspicious circumstances.
    • The Problem Facing the United States• Throughout the United States there may be 40,000 or more skeletal remains stored at medical examiners, coroners and law enforcement agencies that cannot be identified by conventional means.• Few crime laboratories in the United States are equipped to perform the DNA analysis of human remains, especially when they are old or severely degraded.
    • The Problem Facing the United States• 50% or more of the unidentified decedents are most likely homicide victims.• If death was due to a homicide, and the remains disposed of without sample retention, there can be no accountability for the perpetrator.• Advances in DNA technology could make it possible for grieving families to obtain resolution and for those responsible (to) meet justice.
    • University of North Texas Center for Human IdentificationA National Resource for the Identification ofMissing Persons and Unidentified Decedents
    • University of North Texas Center for Human IdentificationThe Center can provide: • A family with information they need to obtain some measure of closure. • Law enforcement with the critical first step ultimately leading to the identification of the perpetrator of the violent crime.
    • 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.
    • www.NamUs.gov
    • NamUs Automatic Searching
    • Forensic Services Available Through NamUs• Forensic Odontologists on staff• Fingerprint Examiner on staff• DNA analyses through the UNT Center for Human Identification’s Laboratory for Molecular Identification• Coordination with local, state and federal DNA laboratories across the country to affect comparisons• Forensic Anthropology through the UNT Center for Human Identification’s Laboratory of Forensic Anthropology
    • Forensic Anthropology
    • Tools to Help Identify Missing Persons and Human Decedents• Forensic Anthropological analysis of human decedents to establish identity and to help determine cause and manor of death• Forensic Odontology and dental identification through comparative analysis of the remains of a decedent and a known persons ante-mortem dental records• Forensic Art to develop a facial post-mortem reconstruction of the decedent, and human aging techniques and technologies
    • Initial Sampling of Bone at Laboratory of Forensic Anthropology Cortical window: left tibia
    • Bone Sample Selection For DNA Analysis• Typically chosen by the anthropologist• Success of STR and mtDNA analysis dependent on the quality of the remains recovered• Prudent selection of sample type will increase the chance of success
    • Compact Bone • Osteocytes are embedded and protected in compact microstructural spaces between concentric layers of bone material • Characteristic of long bones • Femur • Tibiaarthursclipart.org
    • 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
    • Processing samples: Cutting• Sample is moved to the Bone Cutting Lab• Bone Cutting Lab is equipped with 3 custom designed hoods
    • Extraction of DNA from Human Remains
    • Extraction of DNA from Human Remains
    • Extraction of DNA from Human Remains
    • Extraction of DNA from Human Remains
    • DNA Profiles from Human Remains• When human remains are found, they may be in a variety of conditions ranging from recently deceased to fully skeletonized• The approach to obtaining DNA from remains can differ, depending on the state of the remains
    • Remains with Significant Decomposition• Most of the soft tissues will have lost their integrity• Obtaining a DNA profile from liquefied tissues is seldom successful• Bone marrow can sometimes be better preserved and may provide sufficient DNA• If unsuccessful, then recourse should be made to skeletal structures
    • Remains That are Fully Skeletonized• Only hardy structures such as bone, hair, nails and teeth will be available• Skeletal structures, bone matrix and tooth pulp may contain little or no amplifiable nuclear DNA but may be rich in mtDNA• It requires special procedures to release and purify DNA encased within a hardened calcified matrix• The yields of nuclear DNA are typically low and, often, elevated cycle number PCR (>28 cycles) is needed to generate typing results
    • COmbined DNA Index System
    • 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
    • Missing Persons and UHR Data in CODIS as of November 1, 2012At NDIS: 1,081 missing persons profiles 12,445 family reference profiles 6,411 remains profiles 5,978 pedigree trees: 3,216 with more than relative typedUNTCHI’s 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)
    • Challenges Associated With the STR DNA Analysis of Human Remains• Limited amounts of DNA available• DNA samples are often highly degraded• PCR inhibitors often co purify with DNA
    • STR Profiles From Degraded DNA Samples• A multiplex STR amplification reaction can analyse less than 200 pg of DNA, however, the DNA template must be intact where two primers bind as well as between the primers so that full extension can occur• The STR loci with larger sized amplicons in a multiplex amplification are the first to drop out of the DNA profile when amplifying extremely degraded DNA samples
    • PCR Inhibition• Another important challenge to amplifying DNA samples from bones and crime scenes is the fact that the PCR amplification process can be affected by inhibition present in the samples themselves• Samples from crimes scenes and unidentified human remains may contain Taq DNA polymerase inhibitors
    • Examples of Inhibitors that canInterfere with PCR Amplification
    • PCR Inhibition• Inhibitors can: • Interfere with the cell lysis necessary for DNA extraction • Inhibit Taq polymerase activity thus preventing enzymatic amplification of the target DNA• Some inhibitors can co-extract and will remain with the DNA through the entire isolation process
    • Strategies for Dealing with Degraded and Inhibited Samples• Reduction in size of PCR amplicons • Reducing the size of the PCR products will allow amplification from inhibited samples and maximize the chances of recovering information from samples where the DNA is severely fragmented.• Optimization of reaction mix components to facilitate the amplification of inhibited samples • Improvements to buffer systems can provide the ability to overcome inhibitors• Optimization of extraction • Ensuring effective removal of inhibitors • Maximizing recovery of DNA per mg of bone sample
    • DNA Extract from Bone Sample Amplified with Identifiler
    • MiniSTR Development Smaller PCR products work better with low copy number or fragmented DNA templatesConventional miniSTRPCR primer primer STR repeat region miniSTR Conventional primer PCR primer
    • AmpFlSTR MiniFiler ™ Kit
    • Same Bone Sample DNA ExtractAmplified with MiniFiler™ System
    • AmpFℓSTR® Identifiler® Plus• Improved PCR amplification over the current Identifiler kit providing increased sensitivity and robust results in the presence of inhibitors• Improved discrimination for casework samples• Recover more complete DNA data as compared to current version of Identifiler and other STR assay systems• Utilization of the same primer sequences as the original Identifiler® kit and the new Identifiler® Direct Kit eliminating concerns over discordant typing results
    • Bone Sample #1 DNA Extract Amplified with Identifiler
    • Bone Sample #1 DNA Extractnow Amplified with Identifiler Plus
    • NGM System Includes additional Non CODIS miniSTR loci 150 bp 200 bp D10S1048 vWA D16 D2S1338 Amel D8 D21 D18 D22S1045 D19 TH01 FGA D2S441 D3 D1S1656 D12S391
    • Bone Sample Amplified with Identifiler
    • Bone Sample Amplified with the NGM STR System
    • Strategies for Dealing with Optimization of Extraction•Ensuring effective removal of inhibitors•Maximizing recovery of DNA per mg of bone sample•PrepFiler® BTA Lysis Buffer in conjunction with the AutoMate ExpressTM Benchtop Instrument
    • Automated Benchtop Forensic DNA Extraction System• Core reagents are from the PrepFiler™ Forensic DNA Extraction Kit utilizing magnetic particle based nucleic acid purification technology• PrepFiler® BTA Lysis Buffer is a specialized DNA extraction buffer for extracting DNA from calcified tissues (bones, teeth), as well as certain adhesive and paper-containing samples (some cigarettes, tapelifts)• Optimized performance provides comparable or better DNA yields & purity than conventional phenol- chloroform-based methods
    • Automated Benchtop Forensic DNA Extraction System• Fewer tube transfers and greater overall ease of use minimizes the opportunity for sample mix-ups or contamination, while lessening the training burden of new analysts• All required DNA purification reagents are in a sealed disposable plastic cartridge
    • AutoMate ExpressTM Benchtop Instrument • Processes 13 samples in about 30 minutes • Elution Vol: 50 µL • Two protocols (PFLB protocol and BTA protocol) on a single script card Cartridge rackTip and tube rack
    • Cartridge Configuration Universal cartridge suitable for DNA extraction with PrepFiler BTA™ Lysis Buffer be be Tu Tu e n pl io mut SaEl 1 2 3 4 5 6 7 8 9 10 11 12-1 0 Elution solution Lysis buffer Magnetic Wash solution For BTA Particles protocol Isopropanol (500ul)
    • PrepFiler Express BTA™Bones/Teeth Sample Extraction Quantifiler Duo Results Quantifiler Duo Results Human Human 2.0 120 Male Total DNA yield (ng) 100 Total DNA yield (ng) 1.5 80 1.0 60 40 0.5 20 0.0 0 50mg bone 10mg tooth
    • PrepFiler® BTA Lysis Buffer andAutoMate ExpressTM Benchtop Instrument
    • PrepFiler® BTA Lysis Buffer andAutoMate ExpressTM Benchtop Instrument
    • Overnight Demineralization and OrganicExtraction followed by QIAquick Spin Column
    • PrepFiler® BTA Lysis Buffer andAutoMate ExpressTM Benchtop Instrument
    • PrepFiler BTA™ Lysis Buffer Bone Protocol OptimizationIncubation Time + Amount of Bone Powder 25.0 Quantifiler TM Human Total Yield (ng) 20.0 15.0 10.0 5.0 0.0 2h 2h 18h 18h 2h 18h 50m g 50m g 50m g 50m g 200m g 200m g
    • PrepFiler® BTA Lysis Buffer and AutoMate ExpressTM Benchtop Instrument Reproducibilty 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
    • PrepFiler® BTA Lysis Buffer andAutoMate ExpressTM Benchtop Instrument Difficult Older Bone Samples 100 mg bone 100 mg bone 200 mg bone 200 mg bone powder powder powder powder (ng/ul) (ng/ul)H10B-H10C 106.61 Full profile 180.07 Full profileH12B-H12C 1.29E-02 Full profile 4.54E-02 Full profile 018-12 3.61E-03 Partial profile(12) 1.57E-02 Partial profile(17) 029-12 1.13E-02 Partial profile(14) 1.53E-02 Partial profile(23) 0020-12 2.09E-01 Full profile 1.81E-01 Full profile003-14/20 1.32E-02 Partial profile(19) 5.14E-02 Partial profile(23)
    • Sample H10B-10C (100mg)
    • Sample H12B-12C (100mg)
    • 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 Center Fort Worth, Texas USA 817 735-0555arthur.eisenberg@unthsc.edu