Transcript Optimization and Validation of Reduced

DNA Typing and the Development of
Methods for Determination of Degraded
and Compromised Forensic Samples
Bruce R. McCord
Department of Chemistry
Florida International University
[email protected]
www.fiu.edu/~mccordb
The Process of DNA Typing
via the PCR
With 23 pairs of Chromosomes you can get Specific
TPOX
CSF1P0
D3S1358
FGA
D8S1179
THO1 vWA
D5S818
D7S820
D13S317
D16S531
D18S51
D21S11
AMEL
Genotype
Melanie
McCord
The Random Match Probability for this
profile in the FBI Caucasian population
is 1 in 1.56 quadrillion (1015)
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
The Issues
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?
Hundreds of thousands of samples?
Lab Floors like a
Darkroom!
Silver Stained Slab Gel?
Fingers like an Iraqi
voter !
Capillary Electrophoresis
The alternative
1.
Injection, separation, and detection
are automated.
2.
Rapid separations are possible
3.
Peak information is automatically
stored for easy retrieval.
Capillary Electrophoresis
System
Capillary
filled with
entangled
Polymer
Argon Ion
Laser
Capillary
+
Buffer
Deconvoluted
Result
5-20 kV
Buffer
(Sample)
Separation Mechanism
Electrophoretic flow
DNA--
DNA--
DNA--
N
O
O
O
O
O
N
N
N
O
N
N
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
Rg
V
Ogston Sieving
~ 0e-NC
Reptation
 ~1/N
Entanglement
~ f(1/CN)
Right Polymer, Right Voltage means:
Drop Dead Beautiful Results !
Large Multiplex Kits provide Efficient and Rapid
WTC
Disaster
Analysis of Convicted Offender Samples
Jane Doe
231657
But what about degraded DNA ?
Such samples present a
special challenge
Skeletal material
being preped for extraction
DNA Degradation
1. polymer hydrolyzes
(nucleic acids break apart
2. Pyrimidine dimers
(bases X-link)
3. Chemical oxidation
(bases become unreadable)
CH3
O
HO
N H
HO
R
N
O
Thymine glycol
DNA Degradation
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
THO1
CSF1P0
TH01
TPOX
Miniplex 1 vs Powerplex 16
DNA typing of STRs on
microfluidic chips
A genotype in under a minute on a portable
system
Agilent Gene Chip
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
D5
D8
D16
vWA
D18
D13
TH01
CSF
TPOX
FGA
D21
D7
Jane
11,12
10,14
8,12
17,18
13,14
11,13
9.3,9.3
13,13
8,10
24,25
30,32.2
8,9
Daughter LR
12,12
1.465502
10,10
4.950985
8,14
13.72872
17,17
1.776451
13,17
1.887505
10,11
0.736594
9,9.3
1.360359
13,13
10.41341
8,8
0.934981
23,24
1.841485
30,30.2
0.898796
9,12
1.411233
7611.237
A
0.34118
0.10099
0.01821
0.28146
0.13245
0.3394
0.36755
0.09603
0.53477
0.13576
0.27815
0.17715


Likelyhood Ratio =
7,611
Identify confirmed
as a 54 year old
woman missing
since December
2000
Result
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
Less predictable effects
Big Mini
Powerplex 16 9947A Positive
Control
0.250 ng/ 12.5 ul
CSF1PO
TH01
TPOX
FGA
RFU
0 ng
Bone Sample 2003.5.6
0.250 ng/ 12.5 ul
5 ng
10 ng
15 ng
D7S820
The Issue:

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
qPCR Calcium Inhibition
A
B
Control
Lowest inhibitor
concentration
Lowest inhibitor
concentration




No shift in take
off cycle
No change in
melting curve
Efficiency of
amplification
affected
No difference
for size or Tm
Control
Take off cycle
Highest inhibitor
concentration
Highest inhibitor
concentration
C
Control
Lowest inhibitor concentration
Melt
curve
Highest inhibitor concentration
Calcium
Conclusion: Taq Inhibitor
Inhibition of PP16 with CaHPO4
Control Male500pg
CaHPO4 1mM
CaHPO4 1.5 mM
CaHPO4 2mM
CaHPO4 2.7mM
D16, TPOX
D18, CSF, FGA PD, P
qPCR Humic Acid Inhibition
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
Conclusions




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
Acknowledgements

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
Thank you