1 Current and Future tools for DNA Profiling Daniele Podini [email protected]Forensic Science? Current and Future tools for DNA Profiling Daniele Podini [email protected]Forensic Science? Outline • PCR • Forensic Markers • STR analysis • Profile Frequency • Troubleshooting • Analytical Process • Future Technologies THEN THERE WAS PCR Polymerase Chain Reaction PCR • Polymerase Chain Reaction = molecular Xeroxing • “Amplify” the desired DNA fragment(s) • Increased sensitivity • 1988 FBI starts DNA section Dr. Kary Mullis Eccentric Genius 1985 http://www.youtube.com/watch?v=L51UvB5za7c http://www.karymullis.com/pcr.shtml
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Current and Future tools for DNA Profilingforost.org/seminar/Segundo_seminario/Podini-STR-future...Current and Future tools for DNA Profiling Daniele Podin [email protected] Forensic
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The number of consecutive repeat units can vary between people
An accordion-like DNA sequence that occurs between genes
The FBI has selected 13 core STR loci that must be run in all DNA tests in order to provide a common currency with DNA profiles
11 repeats
12 repeats
D5S818
Short Tandem Repeats
11 repeats
12 repeats
D5S818
Short Tandem Repeats
3
11 repeats
12 repeats
D5S818
Short Tandem Repeats The ABI 310 Genetic Analyzer
ABI 310 Genetic Analyzer: Capillary Electrophoresis
Detector Window
11 12
D5S818
Short Tandem Repeats
7 8 9 10 11 12 13 14 15
D5S818
Short Tandem Repeats
11 12
D5S818
Short Tandem Repeats
D8S1179
11 9
4
Scanned Gel Image Capillary Electropherogram
The polymerase chain reaction (PCR) is used to amplify STR regions and label the amplicons with
fluorescent dyes using locus-specific primers 8 repeats
10 repeats Locus 1
8 repeats
9 repeats Locus 2
Multiplex PCR (Parallel Sample Processing)
• Compatible primers are the key to successful multiplex PCR
• STR kits are commercially available
• 15 or more STR loci can be simultaneously amplified
Advantages of Multiplex PCR – Increases information obtained per unit time (increases power of discrimination) – Reduces labor to obtain results – Reduces template required (smaller sample consumed)
Challenges to Multiplexing primer design to find compatible primers (no program exists) reaction optimization is highly empirical often taking months
Statistical estimates: the product rule
0.222 x 0.222 x 2
= 0.1
Statistical estimates: the product rule
= 0.1
1 in 79,531,528,960,000,000
1 in 80 quadrillion
1 in 10 1 in 111 1 in 20
1 in 22,200
x x
1 in 100 1 in 14 1 in 81
1 in 113,400
x x
1 in 116 1 in 17 1 in 16
1 in 31,552
x x
CODIS Short Tandem Repeats
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DNA Profile Frequencies
• The likelihood of two unrelated individuals matching at all 13 loci is less than 1 in 100 trillion (1 X 1012 - 15)
• With 6 billion people on earth you wouldn’t expect to find two matching profiles at 13 loci
• Only identical twins have the same profile
DNA Databasing • DNA Databases allow for ability to search criminal
DNA profiles • 10/13/1998 FBI launched US Database
– Combined DNA Index System (CODIS)
• Revolutionized ability to link crime scene evidence to perpetrators
• Databases effective >60% violent criminals are rearrested within 3yrs. for similar offense
As of February 2011: 9,404,747 offender profiles and 361,176 forensic profiles 138,700 hits assisting in more than 133,400 investigations
Amplification (PCR of STRs) Several Kits Applied Biosystems Profiler Plus Cofiler Identifiler Plus Minifiler Promega Powerplex 16 Powerplex ESX systems
Capillary Electrophoresis Applied Biosystems has monopoly
AFTER EXTRACTION Mixed Samples • ~99% of violent crimes are committed
by men
• DNA Mixtures of male suspect and female victim can pose an analytical challenge, especially when the female contribution is much greater than the male = preferential amplification
• Test for markers found only on the Y-chromosome. Only male DNA is amplified!
Y-STRs
Y-STRs • Paternal relatives all share the same Y-STR
haplotype • 10% of Central Asian males share the same
Y-STR haplotype, thought to belong to Genghis Khan
• Less statistical significance of inclusion • Compared to 13 autosomal STRs • Can increase number of Y- STR loci to increase
Ancestry Informative Single Nucleotide Polymorphisms
AISNPs
Single Nucleotide Polymorphisms (SNPs) that collectively give a high probability of an individual’s ancestry being from one part of the world or being derived from two or more areas of the world.
K.Kidd’s classification
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Phenotype Informative Single Nucleotide Polymorphisms
PISNPs SNPs that provide a high probability that the individual has particular phenotypes, such as a particular skin color, hair color, eye color, etc.
K.Kidd’s classification
Ancestry and Phenotype Inference SNP selection – GWAS
– Anthropology – Human Pigmentation Studies
• Skin • Eye • Hair
– Other
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GWAS • Genome-wide association study:
examination of variation across a genome to identify genetic associations with observable traits and/or specific populations
• Can assay 1 million SNPs at one time (Affymetrix 6.0 array)
• Lots of studies in the last 5 yrs
0
50
100
150
200
250
300
350
2005 2006 2007 2008 2009 2010
Number of GWAS in NHGRI Database from 2005 through December 2010
SNPs with disease associations SNPs with disease associations
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SNPs with disease associations Published Genome-‐Wide Associa4ons through 12/2009,
658 published GWA at p<5x10-‐8 NHGRI GWA Catalog www.genome.gov/GWAStudies
Published Genome-Wide Associations through 6/2010, 904 published GWA at p<5x10-8 for 165 traits
Duffy blood group identifies phenotypes associated with two proteins that appear on the outside of red-blood cells as a receptor
– less skin pigmentation = high /varying polymorphism at MC1R – More skin pigmentation = low polymorphism at MC1R
• Indicates selective pressure over time favors MC1R mutations for less skin pigmentation, while need for more skin pigmentation limited variation in Africa
• Proposed hypotheses regarding the advantages / disadvantages of pigmentation
Effects of UV Radiation on Skin
Image from Jablonski 2004
SNPs
• An initial battery of 105 SNPs has been selected for screening:
Single Base Extension Summary Advantages Disadvantages • Target small amplicons
• Easily multiplexed
• Increased sensitivity
• Equipment already present in Forensic DNA Labs
• Assay design is tedious
The Project: • Sample Collection • Identify DNA markers for ancestry and somatic
traits inference. • Develop and optimize assays to genotype the
selected SNPs. • Perform statistical analyses with the data set to
determine the optimal panel(s) of markers recommended for use in the crime laboratory.
• Develop final panels for casework application. • Disseminate results.
The Project: • Sample Collection • Identify DNA markers for ancestry and somatic
traits inference. • Develop and optimize assays to genotype the
selected SNPs. • Perform statistical analyses with the data set to
determine the optimal panel(s) of markers recommended for use in the crime laboratory.
• Develop final panels for casework application. • Disseminate results.
G/G Homozygotes Eye Color
Dark Blue Blue/Green Dark Green Hazel Light Green Grey Light Blue
Final SNP panel selection Phenotype: eye color HERC 2 - SNP rs12913832
A/A Homozygotes Eye Color
Black/Very Dark Brown
Dark Brown
Light Brown
A/G Heterozygotes Eye Color
Dark Brown Light Brown Hazel Dark Green Blue/Green Light Green Grey
174 Subjects
Homozygous G (69 ~ 40%) clear colored eyes
Homozygous A (56 ~ 32%) brown eyes
heterozygous G/A (49 ~ 28%) present both phenotypes
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Final SNP panel selection Ancestry Duffy - rs2814778 - FY (A-B-)
174 Subjects
91% of homozygous C were African American or African
83% of heterozygous C/T were African American
4% of homozygous T were African American
C/C Homozygote Ethnicity
African
African American Asian
European
C/T Heterozygote Ethnicity
African American Asian
European
Other
T/T Homozygote Ethnicity
African American Asian
European
Hispanic
Other
More work
• Complete optimization of SBE assay • Complete sample collection and analysis • Continue data analysis exploring different
statistical approaches • Complete final SNP panel selection • Final panel development • Optimization on forensic samples
Next Generation Sequencing Technologies
Emulsion PCR
• Fragments, with adaptors, are PCR amplified within a water drop in oil.
• One primer is attached to the surface of a bead. • Used by 454, Polonator and SOLiD.
Bridge PCR
• DNA fragments are flanked with adaptors. • A flat surface coated with two types of primers,
corresponding to the adaptors. • Amplification proceeds in cycles, with one end of each
bridge tethered to the surface. • Used by Solexa.
ABI SOLID
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NGS methods
• Sequencing millions of bases • Low cost per base • Entire genome can be done in a few days • Challenges in data management • Future applications
• At birth sequence entire genome • Personalized medicine • Non human genetics • Forensic Sciences
THANK YOU QUESTIONS? Acknowledgments: Katherine Butler, MS Michelle Peck, BS Jessica Hart, BS Dr Moses Schanfield Dr Pete Vallone NIST Dr Mike Coble NIST Dr James Landers UVA