1
Techniques, Methods and Advances in DNA Testing and Their Impact on g p
Missing Person Investigations
Rhonda K. Roby, MPH, MSProject Coordinator
UNT Center for Human IdentificationUniversity of North Texas Health Science Center
Fort Worth, Texas USA
Techniques, Methods, Advances
SNPs
Electrospray IonizationElectrospray IonizationTimeTime--ofof--flight Mass Spectrometryflight Mass Spectrometry
"One of the greatest satisfactions…is getting the chance to see how a story plays out.
Watching how a new technology evolves after the exciting early days when its potential seems unlimited often provides valuable insights into
Technology
the innovation process."
- David RothmanTechnology ReviewSeptember 2004
2
Lt. Cmdr. Ralph C. Bisz
• Remains repatriated in 1988• DNA testing initiated in 1991• DNA testing multiple times over the nextDNA testing multiple times over the next
17 years• Identified in 2008
Techniques, Methods, Advances
Timeline of forensic genetic analysis
ABO Blood GroupsLandsteiner
DNADouble helix
Watson, Crick
Type II Restriction EnzymesSmith, Wilcox, Danna, Nathans
Multilocus ProbesJeffreys
PCRMullis First PCR
Case
D1S80VNTR
STRsmtDNA Y STRs
Real time PCRDNA Quantification
DNA Working Groups
FBI beginsDNA casework
1900
Red Cell EnzymesSerum Proteins
1953
19701971
SouthernBlotting
1975 1980
VNTRsWyman, White
19841985
First RFLP DNA Case
Jeffreys, Gill et al
1983 1986
1988
DQA1Polymarker Offender DNA
Databases
1997
2004
1987
Automated Sequencer
3
Techniques, Methods, Advances
SNPs
Electrospray IonizationElectrospray IonizationTimeTime--ofof--flight Mass Spectrometryflight Mass Spectrometry
The World Trade Center 9/11/01The World Trade Center 9/11/01
The World Trade Center The World Trade Center 9/11/019/11/01
4
The World Trade Center The World Trade Center 9/11/019/11/01
Challenges•Initially Estimated 0.5
Million to 1.0 Million Specimens
•Extremely Degraded S iSpecimens
•Data Analysis•Management of Data –
Minimally 2.4x109
Bases mtDNA Information
5
Techniques, Methods, Advances
SNPs
Sequencing Capabilities24 hours/day7 days/week1x106 bases/day
The Human Genome
Equivalent to 3% Human Genome/day
Team for mtDNA Processingfor WTC
• Scientists who sequenced the human genome• 1/3 of the team committed to laboratory
processingprocessing• 1/3 of the team committed to quality
control/quality assurance• 1/3 of the team committed to bioinformatics:
LIMS, barcode reading, validation, data analysis
6
Advances
• Robotics• Smaller amplicons for degraded
specimens and higher discriminatory power
• High throughput data analysis• Personal effects for references
Reference Pre-PCR Laboratory
Automation: DNA Re-array and Amplification
7
Tecan Cherry Picking Robot
Sequencing Reaction Set Up
ABI PRISM® 3700 DNA Analyzers
8
MiniFiler™ Kit - mini-STRs
Missing D21, D7, CSF, D16, D2, D18, FGA
D21
D2D16
D18CSF
FGAD7
The Need for MiniThe Need for Mini--STRs for the Analysis STRs for the Analysis of Unidentified Human Remainsof Unidentified Human Remains
Peter Gill and his Colleagues suggest that mini-STR’s are needed:To improve the discriminating power.To improve the sensitivity of testing so that smaller amounts of DNA may be detected.To improve robustness or the quality of the result.
(Forensic Science International 156 (2006) 242(Forensic Science International 156 (2006) 242--244)244)
9
Personal Effect – Razor
Challenges in Missing Person Investigations
• Insufficient Sample Size• Insufficient Quality of DNA• References Available for Comparison
Techniques, Methods, and Advances to Consider
• Collection of Family Reference Specimens• Processing of Family Reference
Specimens• Processing of Evidentiary Material• Advances in Genetic Testing • Advances in Instrumentation• Analysis of Data
10
Family Reference Collection Kits
• Kit Standardization• Evidence receipt• Throughput capabilities increased
Allows for bulk processing• Allows for bulk processing • Amenable to robotics
Swabs
Techniques, Methods, and Advances to Consider
• Collection of Family Reference Specimens• Processing of Family Reference
Specimens• Processing of Evidentiary Material• Advances in Genetic Testing • Advances in Instrumentation• Analysis of Data
11
Tecan Freedom EVO® 100
1. Liquid Handling Arm
7
12. Robotic Manipulator Arm
3. Wash Station4. MagnaBot®5. Te-Shake®
6. Dry Heat Block7. Diluters with
Syringes
2
3
45 6
Processing of Family Reference Samples
800
1000
1200
Family Reference Samples Processed per Year
0
200
400
600
2003 2004 2005 2006 2007
mtDNA +STR STR only
12
Techniques, Methods, and Advances to Consider
• Collection of Family Reference Specimens• Processing of Family Reference
Specimens• Processing of Evidentiary Material• Advances in Genetic Testing • Advances in Instrumentation• Analysis of Data
Processing of Evidentiary Material
Techniques, Methods, and Advances to Consider
• Collection of Family Reference Specimens• Processing of Family Reference
Specimens• Processing of Evidentiary Material• Advances in Genetic Testing• Advances in Instrumentation• Analysis of Data
13
Applied Biosystems310 Genetic Analyzer
Capillary Electrophoresis
ABI PRISM® 3700 DNA Analyzers
Multicapillary Instruments
14
Higher Throughput Multicapillary Instruments
3170xl3170xl
Techniques, Methods, and Advances to Consider
• Collection of Family Reference Specimens• Processing of Family Reference
Specimens• Processing of Evidentiary Material• Advances in Genetic Testing• Advances in Instrumentation• Analysis of Data
● Mini-STRs● Nuclear and mtDNA SNP markers● Alternative methods for rapid
NIJ FUNDS FROM PRESIDENTS’ DNA INITIATIVE
● Alternative methods for rapid sequence analysis
NIJ Cooperative Agreement: 2004-DN-BX-K214
15
MiniFiler™ Kit as an adjunct to the Identifiler® Kit
Missing D21, D7, CSF, D16, D2, D18, FGA
D21
D2D16
D18CSF
FGAD7
Single Nucleotide Polymorphisms (SNPs)
SNPs are single nucleotide basesubstitutions in the genome andaccount for 85% of the geneticaccount for 85% of the geneticvariability in humans.
The Use of SNPs for Human Identification
Why SNPs?• Plentiful – More than 1.8 million SNPs
available in the public/private domain• Highly automatable• Highly automatable• Can be analyzed using a variety of
instrument platforms• Works on highly degraded DNA
• Small amplicons• Cost effective
16
Criteria for SNP Typing Assays
• Easy, Fast, Reproducible• Sensitive• Quantifiable• Multiplexing• Flexible
SNPs as Forensic Markers
Advantages● Abundant
● Small amplicon size
● As small as 45–55 bp - the length of the two PCR primers
● Very useful for severely degraded samples
● Low mutation rate
● ~ 10-8 vs 10-3 for STRs
SNPs as Forensic Markers
Advantages● Bi-allelic nature
● More amenable to automation
● Allele typing interpretation is simpler (e.g., no stutter)
17
mtDNA SNaPshot® Assay17 PLEX (11 HV1 and 6 HV2 SNP’s)
16362 16311 16304162781627016223
16217
1612916126
1609316069
150
195 198200
247
73
1636216311 16304
1627816270
16223 16217 16126
16093
16069150 200 247
73
16223 16129 6 195 198
1636216311
16304
162781627016223
16217 16129
1612616093
16069150
195 198
200247
73
1636216311
16304
16278 1627016223 16217 1612916126
16093
16069150
195 198 200 247
73
Optimization of Sequence Chemistry
Summary of Chemistry Optimization
• Reduced costs• Fewer transfers of DNA evidence• Maintain quality• A bl t b ti• Amenable to robotics• Chemistry available in the future
18
Human Y Chromosome
• Males
• Does not undergo recombination
• The non-recombining region of the human Y chromosme comprises 95% of the chromosome
Y-Chromosome
Rich in repetitive sequences
A number of SNPs haveA number of SNPs havebeen identified
Forensic
BioTrove, Inc OpenArray™ Technology
19
BioTrove Inc. OpenArray™ Plate
• The OpenArray™ plate:
• A unique method to run ~3000 low-volume solution phase assays in parallel
• Equivalent to eight 384-well or thirty-two 96-well plates
• A platform for a wide variety of genomics applications based upon familiar PCR formats
OpenArray™ Process Overview
AutoLoader - Sample loading onto OpenArray™ plate
Automated ESI-TOF of PCR Amplicons on T5000
(M 30H+)30-33- 31-
Measure nucleic acid:Electrospray IonizationTime-of-flight Mass Spectrometry
(M-30H )30
MW = 32,588.90
800 900 1000 1100 1200 1300m/z
29-
27-
25-
35-
37-
39-41-
1089 1093 m/z
20
Mass Spectrometry
● No labeling● Mass accuracy● Multiplexing● Mixture interpretation● Automation
Ibis T5000• Amplicon purification• Automated ESI-TOF analysis
• Robotic arm moves plates for unattended operation• Data analysis• Other functions performed off-line
• DNA extractionDNA extraction• Set up into pre-kitted plates• PCR
• High throughput• 1 well/minute
Mass SpectrometryMass SpectrometryBase Composition
● Number of A, G, C, Ts● Difference in base composition - SNP● Both strands● No post-PCR design
Sample 1 --- A-24, G-30, C-18, T-28
Sample 2 --- A-23, G-31, C-18, T-28A to G transition
21
5000
6000
3285
5.1
3414
3.8
Base composition assignment
32855.1 Da34143.8 Da37058.2 Da37254.2 Da42162.1 Da42710.3 Da
[A40 G9 C40 T19]
3705
8.2
3725
4.2
[A47 G18 C25 T30]
4216
2.1
4271
0.3
[A49 G17 C31 T40]
30,000 32,000 34,000 36,000 38,000 40,000 42,000 44,0000
1000
2000
3000
4000
5000
Mass (Da)
Sig
nal i
nten
sity
Techniques, Methods, and Advances to Consider
• Collection of Family Reference Specimens• Processing of Family Reference
Specimens• Processing of Evidentiary Material• Advances in Genetic Testing • Advances in Instrumentation• Analysis of Data
Expert Systems
22
Techniques, Methods, Advances
SNPs
Electrospray IonizationElectrospray IonizationTimeTime--ofof--flight Mass Spectrometryflight Mass Spectrometry
Summary
• We will continue to evaluate new technology
• We will follow the standards• We will give you• We will give you
our best assess-ment on each case
Acknowledgements
UNTHSCJoseph WarrenGeorge AdamsArthur EisenbergJ h PlJohn PlanzSuzanne GonzalezDixie PetersMelody JosserandChristina Capt