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Penn State Forensics
Science Serving Justice
Establishing an mtDNA Forensic Laboratory
11 August 2006
Mitchell M. Holland, Ph.D.Associate Professor
Biochemistry and Molecular BiologyPennsylvania State University
University Park, PA
Penn State Forensics
Science Serving Justice
Experience
Started the Armed Forces DNA Identification Laboratory (AFDIL) in 1991
Developed methods for mtDNA analysis that have been used for the past 15 years at AFDIL and have been transferred to labs around the world
Developed laboratory methods for the analysis of evidence such as hairs, bone, teeth, and finger nails
Studied various aspects of mtDNA: including apparent mutation rates, heteroplasmy, variant drift, and population diversity
Penn State Forensics
Science Serving Justice
Review Article
Mitochondrial DNA sequence analysis – validation and use for
forensic casework (1999) For Sci Rev 11: 21-50
www.mitotyping.com
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Outline
Blend in the science behind mtDNA analysis with …
Laboratory Design & Practices
mtDNA: Admissibility Issues Contamination? Heteroplasmy? Statistics?
Penn State Forensics
Science Serving Justice
Nucleus
Mitochondria
Nuclear DNA
Mitochondrial DNA
Types of DNA in the Cell
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MtDNA Characteristics
High Copy Number 100’s-1000’s of copies per cell More sensitive test Works on samples that give no STR results
Maternally Inherited Good for historical cases Maternal relatives share your mtDNA profile
Penn State Forensics
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Laboratory Practices for mtDNA Analysis
The sensitivity of mtDNA sequence analysis requires special attention to laboratory design and practices Physical separation for the analysis of evidence
samples (with low quantities of DNA) and reference samples (with high levels of DNA)
Use of hood space Careful handling of evidence Cleaning the surface of the evidence, if possible
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Sources of DNA
Blood Semen Saliva Cigarette
butts Stamps Envelope
flaps
Hat bands Latex gloves Bones Tissue Hair
Anything with Associated Biological Material
Older
Shaft
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Science Serving Justice
Hairs
Hairs are the sample most often tested for mtDNA analysis in criminal cases
Root contains nuclear DNA for STR analysis
Shaft only contains enough mtDNA for analysis
Penn State Forensics
Science Serving Justice
Cleaning Hairs
Hairs are an excellent sample type for mtDNA analysis
The surface of the hair can be “cleaned” with biological detergents
Blood, semen, saliva encrusted hairs or hairs that have been handled can be cleaned to completely remove the surface contributor
Penn State Forensics
Science Serving Justice
Maternal Cousins
Great Grandniece
Deceased
Maternal Inheritance
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Maternal Cousins
Great Grandniece
BrotherDefendant
Maternal Inheritance
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mtDNA Sequence Analysis
Hair Sample
Extraction DNA PCR Amplification
Automated Sequencer Computer Analysis
Sequence Data
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Science Serving Justice
mtDNA Sequence Data
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Science Serving Justice
mtDNA analysis can be performed using different … extraction methods amplification conditions sequencing conditions instruments analysis software
However, when possible the laboratory should use the same general amp/seq methods on both evidence and references
Standardization??
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Human Mitochondrial Genome
0
Displacement Loop or Control region
16000 450
Coding Region
HV1 = 16024-16365 = ~342 bps
HV2 = 73-340 = ~268 bps
HV1 HV2
VR3 = ~16366-72
VR4 = ~341-530
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AGCTTCAGT Human Published Sequence
AACTCCAGC EVIDENCE
Sequence “Profile”
* * *
Established by comparison to apublished mtDNA sequence
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Typical Reported Profile
16189 C16319 T73 G152 T263 G309.1 C
What Does This Mean?
The “C” means that thegenetic code has changedat position 16189 to a “C”
The “309.1 C” means thatan additional “C” is present
after position 309
HV1
HV2
The hair sample was tested usingmtDNA sequence analysis. Thefollowing profile was obtained.
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AGCTTCAGT PUBLISHED
AACTCCAGC EVIDENCE
AACTCCAGC REFERENCE
* * *
MATCH
“Cannot Exclude”Match = Corresponds, Agrees or is Consistent With
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AGCTTCAGT PUBLISHED
AACTCCAGC EVIDENCE
AGCTCCAGT REFERENCE
* **
EXCLUSION
“Exclusions are (generally) Absolute”
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AGCT T CAGT PUBLISHED
AACT C/T CAGC EVIDENCE
AACT C/T CAGC REFERENCE
* **
MATCH
Heteroplasmy = a heterogeneous pool of mtDNA sequences in the cytoplasm of the cell
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Heteroplasmy
AACT C/T CAGC EVIDENCE
AACTCCAGC EVIDENCE
AACTTCAGC EVIDENCE+
Two separate, different sequences present in the sample
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Heteroplasmy v. Contamination
How do you tell the difference between heteroplasmy and contamination?
Heteroplasmy is generally ONLY seen at one position
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Heteroplasmy
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Heteroplasmy v. Contamination
How do you tell the difference between heteroplasmy and contamination?
Heteroplasmy is generally ONLY seen at one position
Heteroplasmy should be reproducible
HOWEVER may show slight drift in variant ratio due to sampling
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AGCT T CAGT PUBLISHED
AACT C CAGC EVIDENCE
AACT C/T CAGC REFERENCE
* **
Match or Exclusion?
Heteroplasmy – “Variant Ratio” Drift
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T
CONTROLTSAR
GEORGIJ ZENIA
The Identification of Tsar Nicholas II and His Family
T/C
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Co-occurrence of Heteroplasmy
TSAR
GEORGIJ
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Bottleneck Theory and Heteroplasmy
Position 16185
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Understanding Heteroplasmy
What is it?
How can you tell the difference between heteroplasmy and contamination? … and show experimentally that you have it?
How is it passed from one generation to the next? … or from one cell to the next??
Which types of samples may have more heteroplasmy than others? … AND WHY??
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Science Serving Justice
Replicates like bacterial cells
When they get too large, they undergo “fission”
The mtDNA is replicated prior to division
Mitochondrion Replication
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mtDNA Variant Drift
C/THeteroplasmy
TT
T
T
C
C
CC Two Mitochondrion
1 with C Homoplasmy
1 with T Homoplasmy
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Hair Biology
GrowthStages
Lasts for0.5-7 years
150 Hairs areshed each day
Human hair histogenesis for the mitochondrial DNA forensic scientist (2001) JFS 46: 844-853
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Hair Biology
Embryonic Development
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Hair Biology and mtDNA
Different hairs may have different ratios of heteroplasmy
Hair mtDNA profiles may or may not share the same ratio of heteroplasmic variants as blood reference samples
Nonetheless, we are usually able to interpret the results and make conclusions
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AGCTTCAGT PUBLISHED
AACTCCAGC EVIDENCE
AGCTCCAGC REFERENCE
* **
“Apparent” Mutational Events
Exclusion or Inconclusive?
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Heteroplasmy Interpretation
At what site does the heteroplasmy occur?
How uncommon is the mtDNA sequence in the population (excluding the position of heteroplasmy)?
How much sequence data do you have?
Are there other reference/exemplar samples that can be tested?
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Results of mtDNA Analysis
In general, an mtDNA result can provide strong circumstantial evidence to associate an evidence sample to an individual
However … issues surrounding the possibility that maternal relatives are associated with the same case should be resolved
mtDNA analysis DOES NOT provide a positive means of identification
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Statistics
What question are you asking?
What question is important to ask in the context of the forensic case?
What databases are you using and are they sufficient to answer the important/forensically relevant questions being asked?
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Databases
Exist for the major population groups As the databases grow in size, more weight
can be placed on the meaning of the match Are there population groups that are under-
represented? What’s the definition of a “group”? Macro v. Micro differentiation of groups There is so much variability within groups that it
isn’t surprising that 100 African individuals sampled from Houston will have different sequence types than 100 from NYC
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Databases
However … Will the “observed frequency” of your mtDNA
sequence in the African population “significantly change” if you use a database from Houston when compared to a database from NYC … or even Nairobi?
The answer is … No
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Statistical Calculations
“Practical” Stats ~6,000,000,000 People in the World ~100,000 mtDNA Profiles Possible Therefore, 1/60,000 Conservative to say … “Can exclude 99% of the
population as the source of a sample using mtDNA” What about “common sequences”? May want to take into consideration the number of
maternal relatives living in the same area of the crime who had an opportunity to commit the crime
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Statistical Methods Confidence Limits from Zero Proportion
For sequences not seen in the database As the database size grows, so does the CLZP
calculation – 99.5% for databases of 500 or more
Normal Approximation of the Binomial For sequences seen multiple time in the database
Bootstrapping For sequence seen very few times in the database
Statistical Calculations
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Courtroom Issues
Did the expert present the results correctly or did the expert mislead the jury/judge? Interpretation of the data Weight of the evidence
Are there issues with respect to the racial background of the defendant in relation to where the crime occurred?
Were the “right” samples tested … or could other samples have been tested?
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Did the laboratory doing the analysis perform the tests correctly?
Does the laboratory have the experience necessary to understand the subtleties of mtDNA analysis? Practices for handling foreign sources of DNA Interpretation of complex data Heteroplasmy – understanding, proper interpretation Was replicate testing performed? … or could it be
performed?
When evaluating the results from an established mtDNA
lab, what’s important?
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Recommend independent review on a case-by-case basis by an outside expert … however, the expert should
Be someone who has a strong understanding of the scientific principles
AND, someone who has considerable practical experience with forensic samples/results
Recommendations
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Science Serving Justice
Thanks for Your Attention
Contact Information
www.forensicdnaconsultants.com
University Address:Penn State University
107 Whitmore Laboratory
University Park, PA 16802
Penn State Forensics
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