DNA Typing and the Development of
Methods for Determination of Degraded and Compromised Forensic Samples
Bruce R. McCord
Department of Chemistry
Florida International University mccordb@fiu.edu
www.fiu.edu/~mccordb

With 23 pairs of Chromosomes you can get Specific
TPOX
D3S1358
CSF1P0
FGA
D5S818
D13S317
D7S820
D8S1179
D16S531
THO1 vWA
D18S51
D21S11
AMEL
Genotype
Melanie
McCord
The Random Match Probability for this profile in the FBI Caucasian population is 1 in 1.56 quadrillion (10 15 )

The Problem:
In the US an average of 115,000 rapes and attemped sexual assaults are reported each year.
Another 250,000 are not reported
There are also
16,500 murders and several million robberies
Rape victim being examined by forensic nurse http://www.ojp.usdoj.gov/bjs/pub/pdf/rsarp00.pdf

1. The current estimated backlog of untested forensic DNA samples is
540,000
2. The number of untested rape kits nationwide is estimated to be 180,000 to
500,000
3. DNA is collected from criminals and data based in a system known as
CODIS

But How To Process All This Data?
Silver Stained Slab Gel?
Lab Floors like a
Darkroom!
Fingers like an Iraqi voter !

1.
Injection, separation, and detection are automated.
2.
Rapid separations are possible
3.
Peak information is automatically stored for easy retrieval.

Capillary Electrophoresis
System
Argon Ion
Laser
Capillary
+
5-20 kV
-
Capillary filled with entangled
Polymer
Deconvoluted
Result
Buffer
Buffer
(Sample)

Electrophoretic flow
--
DNA --
DNA--
N
O
O
N
N
O
O
N
O
N
N
O
PDMA Polymer Structure
(POP4, POP6)

The DNA molecules move through the polymer under the influence of the electric field and are separated low moderate high
Follow the dancing
DNA

R g
V
Ogston Sieving
Reptation Entanglement

~

0 e -NC

~1/N

~ f(1/CN)

Drop Dead Beautiful Results !

Large Multiplex Kits provide Efficient and Rapid
Jane Doe
231657
But what about degraded DNA ?
Skeletal material being preped for extraction
Such samples present a special challenge

1. polymer hydrolyzes
(nucleic acids break apart
2. Pyrimidine dimers
(bases X-link)
3. Chemical oxidation
(bases become unreadable)
CH
3
HO
HO
R
N
O
N
H
O
Thymine glycol

Note loss of intensity of larger alleles
Powerplex 16 9947A Positive
Control 0.250 ng/ 12.5 ul
Bone Sample 2003.5.6
0.250 ng/ 12.5 ul

CSF1P0
1
TH01
TPOX

Agilent Gene Chip
A genotype in under a minute on a portable system

ABI MiniSTRs

Recovery of DNA from degraded Samples
UT Forensic Anthropology Center
Implications for
Mass Disasters
And Questions about
Recovery of Ancient
DNA

Application of Miniplexes to
Casework




Skeletal remains found on April 7, 2002 on the bank of a stream
Caucasian Female, 40-60 years of age
State crime lab would not attempt nuclear DNA due to the fact the body had been in water at some point
A forensic artist produced a sketch based on age ranges, cranial features, and biological profile

Application of Miniplexes



In February of 2003,
DNA extraction and amplification with the
Miniplex sets were performed
Low amounts of DNA yielded a profile for 12
CODIS loci covered by the Miniplexes
Profile information was given to the forensic anthropologist and the coroner’s office


October, 2004: the coroner was contacted by a woman who had seen the sketch on the internet and thought it looked like her mother
Buccal swabs from the suspected daughter were taken and sent to the McCord lab at FIU

Application of Miniplexes
Locus Jane
D5 11, 12
Daughter LR
12 ,12
A
1.465502 0.34118
D8
D16
10,
8
14
,12
10
8
,10
,14
4.950985 0.10099
13.72872 0.01821
vWA
D18
D13
17 ,18 17 ,17
13 ,14 13 ,17
11 ,13 10, 11
1.776451 0.28146
1.887505 0.13245
0.736594 0.3394
TH01
CSF
9.3
,9.3
9, 9.3
13 ,13 13 ,13
TPOX 8 ,10
FGA 24 ,25
8 ,8
23, 24
D21
D7
1.360359 0.36755
10.41341 0.09603
0.934981 0.53477
1.841485 0.13576
30 ,32.2
30 ,30.2
0.898796 0.27815
8, 9 9 ,12 1.411233 0.17715
7611.237


Likelyhood Ratio =
7,611
Identify confirmed as a 54 year old woman missing since December
2000

Identified as Roberta Gile, Age 54
Missing since December, 2000

Application of
MiniSTRs in bone/bone reassociation
Yugoslavia
Parsons et al, Forensic Science
International: Genetics 1
(2007) 175–179

The Problem of Degradation vs Inhibition in DNA typing
Degraded DNA Sample Humic Acid Inhibited DNA Sample
Ski slope effect
Powerplex 16 9947A Positive
Control 0.250 ng/ 12.5 ul
Less predictable effects
Big Mini
TH01
TPOX
CSF1PO
FGA
D7S820
0 ng
5 ng
Bone Sample 2003.5.6
0.250 ng/ 12.5 ul
10 ng
15 ng


With increasing interest in the forensic community in the interpretation of compromised samples and mixtures, we need to be able to better interpret electropherograms in court




We need to determine the relative effects of DNA degradation and inhibition on peak height ratios.
We need to understand the combinatorial effects of different inhibitors
We need to understand the environmental aspects of degradation and soil inhibition
We need to explore the interpretation of low level mixtures in the presence of a major contributors

B A
Control
Lowest inhibitor concentration
Control
Lowest inhibitor concentration
Take off cycle




No shift in take off cycle
No change in melting curve
Efficiency of amplification affected
No difference for size or Tm
C
Highest inhibitor concentration
Lowest inhibitor concentration
Melt curve
Highest inhibitor concentration
Control
Highest inhibitor concentration
Calcium
Conclusion: Taq Inhibitor

Inhibition of PP16 with CaHPO
4
Control Male500pg
4
CaHPO
4
1.5 mM
CaHPO
4
2mM
CaHPO
4
2.7mM
D16, TPOX D18, CSF, FGA PD, PE

Take off cycle




Shift in take off cycle
Change in melting curve
No efficiency of amplification change
Size effects on melt curve
Melt curve
Conclusion: Sequence specific Inhibitor

Inhibition of PP16 with Humic Acid
Control Male500pg
Humic Acid
16ng/uL
Humic Acid
24ng/uL
Am D3 d18 CSF PE PD





The key to automated forensic DNA typing was multiplex PCR amplification with capillary gel electrophoresis with laser induced fluorescence
Redesign of PCR primers using MniSTRs permits recovery of badly degraded DNA
Mechanisms for understanding DNA degradation can be determined using real time
PCR.
Technology transfer: multiplex capillary electrophoresis, miniSTRs, realtime PCR technologies impact peoples lives. For example:this year the incidence of reported rape has hit a 20 year low


Funding





National Institute of Justice
National Science Foundation
J Edgar Hoover Foundation
Federal Bureau of Investigation
TSWG

Collaborators






John Butler, NIST
Eric Buel and Jan Nicklas,
Vermont Forensic Laboratory
George Duncan, BSO Crime Lab
Ira Lurie, DEA
Sonja Rawn, OSFM Forensic Lab
Kelly Mount FBI

Researchers

Yin Shen
Jiri Drabek
Maximilien Blas
Maribel Funes
Silvia Zoppis
Kerry Opel
Denise Chung
Maurice Aboud
Heather LaSalle
Tanya Madi
Robyn Thompson
Brittany Hartzell
Oscar Cabrices
Stefano Boulas
William Kennedy