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

arthur.eisenberg@unthsc.edu