Overview of Rapid DNA Te

2009 Biometric Consortium Conference 

September 23, 2009 

Dr. Peter M. Vallone 

Biochemical Science Division 

National Institute of Standards and Technolo
Outline 

• Basics of DNA Typing • Rapid PCR 
Basics of Forensic DNA Tes
General Characteristics of Genomic 

• Each person has a unique DNA 

(except identical twins) 

• Each person's DNA is the same 

cell (DNA from skin cells will match D

blood cells) 

• An individual’s DNA profile rema

same throughout life 

• Half of your DNA comes from yo

mother and half from your fathe
Forensic DNA Testing 

Probe subsets of genetic variation in ordedifferentiate between individuals DNA typing must be done efficiently and reproducibly (information must hold up in Typically, we are not looking at genes – litinformation about race, predisposal to diseor phenotypical information (eye color, hehair color) is obtained 
Sources of Biological Eviden

• Blood• Semen• Saliva• Urine• Hair• Teeth• Bone• Tissue

Bl

s

best results with >100 cells, but DNA profiles c

recovered from as little as a single cell 
Applications 

• Forensic cases: matching suspect with evid• Paternity testing: identifying father • Missing persons investigations • Military DNA “dog tag” • Convicted felon DNA databases • Mass fatalities: putting pieces back together • Historical investigations • Genetic genealogy • DNA as a biometric tool 
DNA Testing Requires a Reference S

A DNA profile by itself is fairly useless because it has no context... DNA analysis for identity only works by comparison – you need a reference sample 

Crime Scene Evidence compared to Suspect(s) (Forensic Cas

Child compared to Alleged Father (Paternity Case) Victim’s Remains compared to Biological Relative (Mass DisaSoldier’s Remains compared to Direct Reference Sample (Ar
Technology 1.5 h 

Steps in Forensic DNA Analys

Usually 1-2 day process (a minimum of ~8 hours) 

DNA 

Extraction 

Multiplex PCR Amplification 

Sample Collection 

& Storage 

Blood Stain Buccal swab

DNA 

Quantitatio

Statistics Calculated 

DNA Database search 

Biology

~3.5 h 

1.5 h 1.5 h 

Paternity test 

Reference sample 

Applied Use of Information 

DNA separation and sizin

Genetics

STR Typing 

Interpretation of Res
AMEL D5S818 

D13S317

D16S539 D2S133

D18S51

TPOX VWA D19S433

D3S1358 TH01

Identifiler (Applied Biosystems) 15 STR Loc

Information is tied together with multiplex PCR and data 

D21S11 

D8S1179 D7S820 CSF1PO 

(15,16)-(29,29)-(9,11)-(10,11)-(16,17)-(6,7)-(8,12)-(10,11)

(14,16)-(15,17)-(8,12)-(11,15)-(X,Y)-(9,11)-(19,22

The Random Match Probability 

(RMP) is over 1 in 800 trillion 

for unrelated individuals 

The chance of someone else having this exact sam

This test contains the 13 FBI core loci (NDIS

FGA 
Recent Work with Rapid P

At NIST we are working on new PCR me

to reduce the time for PCR down to 20 m

Polymerase 

Chain 

Reaction 

(PCR) 

Is a means to create billions of 

exact copies of the human 

genome – necessary/essential 

for DNA typing Multiplex PCR 

~3.5 h 
Why go Faster? 

Applications for Rapid PCR 

• Integrated devices (‘Lab on a Chip’) • Screening at a point of interest (airport, border

scene, intelligence community) 

• Rapid STR typing ‘in the field’ 

– Potential for situations/cases when a quick result is – Provide initial screening information 

• Decrease overall time required for STR typing 

DNA as a Biometric tool 
Current Efforts Towards 

Portable/Mobile DNA Devic

• Network Biosystems (Woburn, MA

http://www.netbio.com 

• MicroLab Diagnostics and Lockhee

Martin (Charlottesville,VA) 

http://www.microlabdiagnostics.com 

• Microchip Biotech (Dublin, CA) 

http://www.microchipbiotech.com 
Goals for Rapid DNA 

Typing Platforms 

• Create an integrated system capab

taking a swab and perform DNA tein approximately 1 hour 

• Little user interaction (or experienc• Rugged Swab in...answer out

• Robust • Simple data interpretation 
Can novel assays be developed to further 

understanding/limits of rapid multiplex PC

Sample 

Extraction 

~2 h 

Quantitation 

~1.5 h 

PCR 

~3 h 

CE Ru

~1.5 h 

Typical STR Typing Workf

Can the time required for PCR thermal cycling be r

Alter thermal cycling parameters 

Evaluate faster polymerases 

Evaluate faster thermal cyclers 

Test commercial STR typing kits 

Goal: cycling in less than 40 minute

Trying simple things first... 
Thermal Cyclers 

Cepheid SmartCycler Purch

Ramp rate = 10

oC/s fundi

Eppendorf 

Mastercycler pro 

Ramp rate = 6

oC/sec 

Applied Biosystems 9700

Ramp rate = 4

oC/s 
36 Minute PCR Amplification 

on AB 9700 Cycler 

28 cycles, Identifiler STR k
20 Minute PCR Amplification 

on Cepheid Cycler 

28 cycles, Identifiler STR k
19 Minute PCR Amplification 

on Eppendorf Cycler 

28 cycles, Identifiler STR k
Rapid PCR Article 

Vallone, P.M., Hill, C.R., Butler, J.M. (2008) Demonstration of rapid 

amplification involving 16 genetic loci. FSI Genetics 3(1): 42

Rapid Amplification of Commercial STR Typing Kit

Presented at the International Society of Forensic Genetic

meeting in Buenos Aires Argentina (September 16, 20

(Voted Best Poster Presentation) 
Rapid PCR Summary 

• Rapid multiplex PCR amplification is po

– Compatible with commercial STR typing kit– Provides same genotypes as standard cycl

• Fast (optimized) polymerases are neede• Further work 

– Applying techniques to integrated platforms – Formal validation of technique – Sharing results with PCR community – Understanding the kinetics of PCR 
Thank you for your attenti

Questions? Peter.Vallone@nist.gov 301-975-4872 

Outside funding agencies: FBI - Evaluation of Forensic DNA Typing as a Biometric Tool NIJ – Interagency Agreement with the Office of Law Enforcem