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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
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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
The UNT Center for Human Identification
3 Divisions: • Laboratory of Forensic
Anthropology • Laboratory for Molecular
Identification • Forensic Services Unit / NamUs
Focus: • 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.
• 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.
The Problem Facing the United States
University of North Texas Center for Human Identification
A National Resource for the Identification of Missing Persons and Unidentified Decedents
The 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.
University of North Texas Center for Human Identification
Free. Secure. Nationwide.
www.namus.gov
The National Missing and Unidentified Persons System offers law enforcement agencies, medical examiners, coroners, family members and victim advocates a powerful tool for resolving missing and unidentified persons cases.
www.NamUs.gov
NamUs Automatic Searching
• 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 Services Available Through NamUs
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
• 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
Bone Sample Selection For DNA Analysis
Compact Bone
• Osteocytes are embedded and protected in compact microstructural spaces between concentric layers of bone material
• Characteristic of long bones • Femur • Tibia
arthursclipart.org
What Human Remain Samples Should be Submitted?
• 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)
Bones: most preferred to least preferred
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, 2012
At 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 typed UNTCHI’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 can Interfere with PCR Amplification
• 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
PCR Inhibition
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
STR repeat region miniSTR primer
miniSTR primer
Conventional PCR primer
Conventional PCR primer
Smaller PCR products work better with low copy number or fragmented DNA
templates
AmpFlSTR MiniFiler ™ Kit
Same Bone Sample DNA Extract Amplified 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 Extract now Amplified with Identifiler Plus
NGM System Includes additional Non CODIS miniSTR loci
D10S1048 vWA D16 D2S1338
Amel D8 D21 D18
D22S1045 D19 TH01 FGA
D2S441 D3 D1S1656 D12S391
150 bp 200 bp
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 rack
Tip and tube rack
Cartridge Configuration
1 2 3 4 5 6 7 8 9 10 11 12Sam
ple Tub
e
Elution
Tube
0-1
1 2 3 4 5 6 7 8 9 10 11 12Sam
ple Tub
e
Elution
Tube
0-1
Magnetic Particles
Isopropanol
Wash solution Elution solution
Universal cartridge suitable for DNA extraction with PrepFiler BTA™ Lysis Buffer
Lysis buffer For BTA protocol (500ul)
PrepFiler Express BTA™ Bones/Teeth Sample Extraction
Quantifiler Duo Results
0.0
0.5
1.0
1.5
2.0
50mg bone
Tota
l DN
A y
ield
(ng)
HumanMale
Quantifiler Duo Results
0
20
40
60
80
100
120
10mg tooth
Tota
l DNA
yie
ld (n
g)
Human
PrepFiler® BTA Lysis Buffer and AutoMate ExpressTM Benchtop Instrument
PrepFiler® BTA Lysis Buffer and AutoMate ExpressTM Benchtop Instrument
Overnight Demineralization and Organic Extraction followed by QIAquick Spin Column
PrepFiler® BTA Lysis Buffer and AutoMate ExpressTM Benchtop Instrument
PrepFiler BTA™ Lysis Buffer Bone Protocol Optimization
Incubation Time + Amount of Bone Powder
0.0
5.0
10.0
15.0
20.0
25.0
2h 50mg
2h 50mg
18h 50mg
18h 50mg
2h 200mg
18h200mg
Qua
ntifi
lerTM
Hum
an T
otal
Yie
ld (n
g)
PrepFiler® BTA Lysis Buffer and AutoMate ExpressTM Benchtop Instrument
Reproducibilty
Quantification (ng/ul)
Total DNA yield (ng)
200mg Automate 1 4.22E-02 2.11
200mg Automate 2 4.70E-02 2.35
200mg Automate 3 4.69E-02 2.34
200mg Automate 4 4.58E-02 2.29
PrepFiler® BTA Lysis Buffer and AutoMate ExpressTM Benchtop Instrument
Difficult Older Bone Samples 100 mg bone
powder (ng/ul)
100 mg bone powder
200 mg bone powder (ng/ul)
200 mg bone powder
H10B-H10C 106.61 Full profile 180.07 Full profile
H12B-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 profile
003-14/20 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 Genetics Co-Director UNT Center for Human Identification,
Institute of Investigative Genetics University of North Texas Health Science Center
Fort Worth, Texas USA 817 735-0555