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Outline
Identification Classification Individualisation
Methods of Identification Development of Forensic DNA Forensic issues Interpretation of results Future
Identification
Classify – Eg. Human or not? Human – Sex? Race? Age? Identification – Nationality? Identification – Locality? Identification – Identity
(Individualisation!)
Human Identification
How do we identify an individual?
The human brain associates a name with an individual using a variety of information including physical appearance, mannerisms, voice and speech, etc.
Visual Identification
Despite the complexity and accuracy of visual identification of an individual, it is well known that mistakes are common and in some circumstances unreliable and difficult.
Unreliable visual ID
Time factors – aging, etc. Psychological and emotional factors –
poor recall Lighting – inadequate or problematic
lighting Physical changes especially in forensic
work.
Other physical attributes
Ear-shapes Lip prints Retinal scans Voice patterns (?) Handwriting (under scrutiny!)
Development of DNA Fingerprinting 1985, Sir Alec Jeffries, described
variable number tandem repeats (VNTR`s) and developed the technique restriction fragment length polymorphisms (RFLP)
Briefly, it used restriction enzymes to cut the regions of the DNA surrounding the VNTR’s.
History
First use in casework in the U.K. in 1985.
First commercial labs in the U.S. in 1986
Used by the FBI in the U.S. in 1988. Used in Hong Kong in early 90’s. DNA-PCR technology used in Hong
Kong in 1997.
Uniqueness
Except for identical twins the DNA of an individual is unique.
The number of different chromosomes that a child receives from parents are 246
Requirements for forensic casework Reliability of technique Reproducible results No laboratory error Security of test samples and results
DNA Polymorphisms
Sequence polymorphism….AGACTAGACATT…..
….AGATTAGGCATT…..
Length polymorphism
….AATGAATGAATG….
….AATGAATG…..
RFLP
Restriction fragment length polymorphism
A restriction enzyme is used to cut the DNA into fragments at specific points
These fragments of different lengths are separated by an electric current. The fragments of interest are then radiolabelled by hybridisation.
RFLP
Many RFLP systems are based on change in a single nucleotide. They are said to be diallelic
Thus only two common alternative forms and three phenotypes, two homozygous and one heterozygous.
PCR Based Systems
Length Polymorphisms STR kits - Short Tandem Repeats
Sequence Polymorphisms PolyMarkers eg. DQ-alpha/A1
(HLA - DNA) Mitochondrial DNA
Automated systems now available.
STR
Short-tandem repeats Higher incidence of homozygotes, since
the system is less polymorphic Several loci can be amplified Increase discriminating power with use
of multiple probes.
Length Polymorphisms
PCR used to amplify this. Much simpler than RFLP analysis
because the DNA of interest already amplified.
Bands stained directly Trend towards fluorescent detection and
automated analysis.
Forensic use of DNA technologies Identification of small quantities of
biological samples found, e.g. blood stains, semen, saliva stains, etc.
Differentiation between origins of samples found.
Linking and/or grouping of unknown sample.
Obstacles in forensic casework Small quantity of samples to work with. Contamination of samples. Poor preservation of material from
which DNA is to be extracted. Eg. Contaminated stains, decomposition of tissues.
Complicating factors and forensic challenges Multiple contributors (sources) – mixed
sample. Differential extraction Degradation Contamination
Inhibition of enzymes Non-human DNA
CODIS
Combined DNA Index System 1990 as a pilot project at the FBI
Laboratory. Now in more than 100 public
laboratories
Quality and standards
DNA Advisory Board Quality assurance standards
Laboratory Validation American Society of Crime Lab Directors
Laboratory Accreditation Board European DNA Profiling Group Interpol European Working Party on DNA
Profiling
Controls
Monitoring for False Negatives False negatives arises from inhibitors.
Safeguard against false negatives Positive controls – similar physiochemical
properties and contains known DNA.
Controls Monitoring for False Positives False positives generally arises from
contamination. Safeguards against false positives
A negative control – undergoes the whole procedure, similar physiochemical properties except for genetic property.
A blind control – undergoes all extraction, purification and amplification procedures, except that it does not contain any sample material.
A no-template control – serves only for the amplification reagents and conditions. It contains every amplification reagents except DNA
Significance of results
Three possible conclusions:- 1. Exclusion – they are different. 2. Inconclusive 3. Similar
Similarity
Three possible scenarios:- 1. Sample from a common source 2. Coincidence 3. Accident (Error)
Frequency Estimate Calculations Hardy-Weinberg equilibrium
There is a predictable relationship between allele frequencies and genotype frequencies at a single locus. This is a mathematical relationship that allows for the estimation of genotype frequencies even if the genotype has not been seen in an actual population survey.
Frequency Estimate Calculations Linkage equilibrium
Defined as the steady-state condition of a population where the frequency of any multi-locus genotypic frequency is the product of each separate locus. This allows for the estimation of a DNA profile over several loci, even if the profile has not been seen in an actual population survey.
DNA evidence in Court
This posed a problem due to a hosts of poorly researched and performed work and estimate calculations.
It is now quite widely accepted and increasingly the frequencies are individualising e.g. 1 in several billions!!
Population Data
Population data is required to obtain the various frequencies of occurences.
Systems used require careful validation prior to use and also require internal and external controls for each test.
Future - Now
Automation DNA databases Variant Repeats – approaches individualisation
with just one or two loci More loci and marker systems SNP’s Quality control will continue to be an area of
contention. More applications and more probes Faster automation, etc
DNA Databases
Privacy issues Quality control of data Convicted samples vs. forensic case
work samples
Ancient DNA
Made possible by the availability of PCR The term ancient DNA now covers any bulk
or trace DNA from a dead organism or parts of it, as well as extracorporeally encountered DNA of a living organism.
Therefore any DNA that has undergone autolytic or diagenetic processes or fixation is considered “aDNA”
Mitochondrial DNA Advantage of having multiple copies present;
therefore higher chance of detection in degraded material compared with nuclear DNA.
Increasing use, different methodologies. About 4800 human mtDNA sequences available
in forensic databases. An observed sequence is reported as the number
of times an observed sequence is present in known databases and as such a frequency percentage is given. Possible to exclude 99%.
Mitochondrial DNA
Discriminatory powers are still poor. Similarities may be seen in several
individuals with similar maternal root. Useful in “population genetics” and in
tracing the maternal line.
Y-Chromosome
Increasing interest since it was sequenced. Y-chromosomes had about 6 of its 50 million DNA letters in palindromes.
Have also been used to trace the “male line” in population studies
Useful for working on mixed samples. May be key to “racial or regional characterisation”.
Large numbers of commercial kits now available.
Future
Low-copy numbers – risk of contamination high
Racial – regional origins. Physical attributes from DNA Psychological/behavioral attributes Genetic predisposition for drug
tolerance, for rare disorders, etc.
Recommended reading DNA Technology in Forensic Science – National Research
Council (1992) ISBN 0-309-04587-8 The Evaluation of Forensic DNA Evidence – National
Research Council (1996) ISBN 0-309-05395-1 An Introduction to Forensic DNA Analysis – K. Inman & N.
Rudin. CRC Press (1997)ISBN 0-8493-8117-7
Forensic DNA Typing – Biology and Technology Behind STR Markers - John M. Butler. Academic Press (2001) ISBN 0-12-147951-X
Recommended reading
Rutty GN et al “Contamination of mortuary instruments and work surfaces by human DNA; A significant problem in forensic practice?” Int. J Legal Med 2000; 114: 56-60
B.S. Weir “Population genetics in the forensic DNA debate.” Proc. Natl. Acad. Sci. USA Vol 89 pp 11654-11659, December 1992
John Whitfield “Y chromosome sequence completed” Nature Science Update 19 June 2003.