Techniques, Methods and Advances in Missing Person ......Polymarker Offender DNA Databases 1997 2004 1987 Automated Sequencer 3 Techniques, Methods, Advances SNPs Electrospray Ionization

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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


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

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SNPs


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

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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

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Tecan Cherry Picking Robot

Sequencing Reaction Set Up

ABI PRISM® 3700 DNA Analyzers

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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)

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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

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Family Reference Collection Kits

• Kit Standardization• Evidence receipt• Throughput capabilities increased

Allows for bulk processing• Allows for bulk processing • Amenable to robotics

Swabs




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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




Processing of Evidentiary Material



Specimens• Processing of Evidentiary Material• Advances in Genetic Testing• Advances in Instrumentation• Analysis of Data

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Applied Biosystems310 Genetic Analyzer

Capillary Electrophoresis

ABI PRISM® 3700 DNA Analyzers

Multicapillary Instruments

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Higher Throughput Multicapillary Instruments

3170xl3170xl



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

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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

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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)

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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

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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

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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

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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

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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




Expert Systems

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SNPs


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

Documents

Techniques, Methods and Advances in Missing Person ......Polymarker Offender DNA Databases 1997 2004 1987 Automated Sequencer 3 Techniques, Methods, Advances SNPs Electrospray Ionization