Alec Jeffries: DNA As A Forensic Tool

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Transcript Alec Jeffries: DNA As A Forensic Tool

Alec Jeffries: DNA as a forensic tool
 Colin Pitchfork case: First time DNA was used in a legal setting
 1983 and 1986: two attacks and murders of 15 year old girls,
several years apart, very similar.
 14 year old Richard Buckland confesses to the crime of Dawn
Ashworth, but maintained innocence regarding Lynda Mann’s
murder.
 Police sought out Dr. Jeffries help to connect Buckland to both
crimes. Jeffries confirms that same man committed both crimes,
but not Richard Buckland.
 5000 males are tested for blood type and DNA profile. Colin
Pitchfork is eventually convicted of both crimes. Buckland is
exonerated.
DNA evidence prior to 1985
Scientists knew that if small changes were made to
DNA, quite often the offspring would not be viable.
Even small changes in an amino acid sequence,
dramatically effects the shape and function of a
protein – which may be deadly.
This led to many people believing that all of our DNA
was almost identical to each other, not much variation
from person to person.
DNA basics: Review
 DNA is made up of nucleotide building blocks,
that are connected in chains
 Each nucleotide has three parts:
 Phosphate group
 Deoxyribose (sugar)
 Nitrogenous base
The Nitrogenous bases hang off the phosphate/sugar
backbone and meet in the middle (rungs of a ladder)
 DNA strands can be up to 6 billion nucleotides, connected by
hydrogen bonds
 The DNA double strand that we are used to seeing, is created
through hydrogen bonds that are organized in a very specific
way:
 Because of the structure of the four nitrogenous bases:
Adenosine, Cytosine, Guanine, Thymine: hydrogen bonds can
only form between certain pairs
 Electrostatic Attraction only exists between Adenosine -Thymine
and Cytosine-Guanine.
 If Thymine meets Cytosine – they would repel each other, and no
hydrogen bond would be formed.
 Terminology:
 Genes: a particular sequence
of nucleic acids that code for
something
(proteins/enzymes)
 Strand of DNA: the genetic
and nonsense lengths of DNA
 Chromosome: a long chain of
DNA – part of the entire DNA of
a cell
 Genome: all of the
chromosomes
 Chromosomes are formed when DNA coils itself around
histone proteins in the nucleus or mitochondria.
 It is a way to package genetic material into more
manageable units.
 For humans: we have 23 pairs of chromosomes – each
with their own unique shapes and sizes.
 The X and Y chromosomes determine biological gender
– Females having XX and males having XY.
 There are some cases where the sex chromosomes
aren’t so cut and dry:
 Klinefelter’s Syndrome – XXY. It is thought that about 1
in 500 males have an extra X chromosome, but many
don’t have any symptoms.
 47, XYY syndrome: an extra Y chromosome in each cell..
About 1 in 1000 – no abnormal physical development is
usually associated.
 Triple X: girls with an extra X chromosome, 1 in 1000 –
usually tall and mild learning disabilities
 Turner Syndrome: about 1 in 2500 girls – missing part
of the 2nd X chromosome. Usually infertile, shorter, mild
learning disabilities.
 Genes are a small region that are the code or
instructions on the synthesis of a molecule – usually a
protein or an enzyme.
 The unique arrangement of the “letters” – the nucleic
acids – dictates how to prepare these molecules.
 Where the gene is located on the chromosome is called
the locus. When we talk about more than one – loci.
 Alec Jeffries discovered something related to how genes
were mapped into strands of DNA…
 Genes are short coding regions of DNA, associated with
certain start and stop sequences that contributed to
genes being turned on and off – proteins made etc.
 Genes are separated by long regions of DNA that are
non-coding regions – junk DNA – nonsense strands.
 Sometimes these non-coding regions are also called
hyper variable.
 These regions are extremely different from person to
person – the length of the non coding regions is highly
individual.
 In 2003: Human Genome Project
completed: mapped entire genome:
every single base sequence for every
gene.
 Discovered that about 95% of the DNA
in human cells is the same as the DNA
of a fruit fly.
 Only about 1.5% of DNA codes for
biological compounds, necessary for life.
 The amount DNA that doesn’t code for
anything is far greater than the DNA
that codes for specific molecules of life.
The Junk DNA is what we analyze to
look for difference from person to
person.
DNA: From Genes to Proteins
 Two processes: transcription and translation are
responsible for taking biochemical code and turning
them into proteins and other molecules responsible for
visible traits.
 Transcription: the process by which DNA is converted
into RNA
 Translation: the process by which RNA produces proteins
Transcription Basics:
 Almost 50 different proteins and transcription factors
recognize and bind to promoter sites
 An enzyme, RNA polymerase binds to the complex of
transcription factors and this opens up the double helix
(breaking the hydrogen bonds)
 This allows the strand to become accessible for base pairing
the appropriate RNA nucleotides.
 RNA Polymerase works by reading the DNA in a 3’  5’ direction
 3’ and 5’ prime refers to the carbon atoms in the sugar
molecules that are the DNA backbone.
 The 3’ carbon is bonded to hydroxyl group (OH)
 The 5’ carbon is bonded to the phosphate group (PO4)
 As RNA polymerase moves down the chain of DNA, it assembles
ribonucleotides using the following rules:
 A binds with Uracil
 T binds with A
 G –C still base pairs
 The RNA that is created is synthesized in the the 5’ to 3’
direction.
 http://vcell.ndsu.nodak.edu/animations/transcription/mo
vie-flash.htm
 http://www.stolaf.edu/people/giannini/flashanimat/molg
enetics/transcription.swf
Translation: RNA  Proteins
 Unlike DNA, RNA is able to exit out of the pores of the
nucleus.
 Three types of RNA involved in translation:
 mRNA – carries that message from DNA
 rRNA – works to create the ribosome for protein production
 tRNA – reads the mRNA and delivers the corresponding
amino acids
 As the mRNA leaves nucleus, moves
through the cytoplasm to the rough
endoplasmic reticulum which
contains ribosomes.
 Each group of three mRNA bases is
called a codon.
 The tRNA is able to recognize mRNA
codons, and match them up to a
particular amino acids. The tRNA
serves as an adaptor between the
mRNA and the amino acids.
 Each tRNA possesses an anticodon: a
unique sequence that matches up with
the mRNA codon through base pairing.
 Two things have to happen:
 The tRNA has to carry the appropriate
amino acid
 The codon- anticodon base pairing must
occur
 There are 64 codons in mRNA, 61 of them
code for an amino acid.
 The other three are stop codons which
signal the tRNA and the ribosomal subunit
to stop translation.
 Translation is always initiated with AUG
codon – which codes for methionine.
 Bacteria cells (prokaryotes) use other codons
besides AUG – but always result in methionine.
 In eukaryotes, ribosomal subunit first binds to a
7methylguanosine cap at the 5’ end and then
scan down until the AUG codon is recognized.
Translation
 Initiation: many ribosomal subunits bind together and
form an initiation complex.
 Elongation: The ribosome unit has several regions where
different tRNA can bind and in a highly regulated
mechanism tRNA anticodon and codons bind and amino
acids are strung together peptide bonds.
 This occurs until the ribosome reach's the stop codon,
and the tRNA molecules are released and the ribosomes
subunit / mRNA dissociate.
Protein Synthesis Animations
 http://highered.mcgrawhill.com/sites/0072507470/student_view0/chapter3/ani
mation__how_translation_works.html
 http://www.stolaf.edu/people/giannini/flashanimat/molg
enetics/translation.swf
 http://www.biostudio.com/demo_freeman_protein_synth
esis.htm
 The amino acid sequence is known as the primary
structure – it is held together with peptide bonds.
 As the amino acids begin to string together, their
different molecular polarities, shapes, sizes,
result in intermolecular attractions, interactions.
 This causes the chain to fold in different ways,
which is their secondary structure
 Alpha Helix: tight coil
 Beta Pleats: two strands side by side
Finally, tertiary structure is achieved with
the help of proteins called chaperonins –
which modify the secondary structure.
The final shape of the protein determines
it’s function. (Remember the lock and key
model of the enzyme)
Prions such as the Mad Cow disease cause
damage by manipulating the structure and
therefore function of proteins in the brain.
Forensic Applications of DNA
 Tandem Repeats are also known as microsatellites
 These are short repeated nucleotide sequences. In
humans there are about 100,000 blocks of “CA”
 The number of CA repeats at a locus varies
between individuals and can therefore be used as a
genetic marker.
 Almost 50% of the genome are these repeating
sequences – short sequences that are repeated
back to back.
 Of particular interest in forensics is Variable
Number Tandem Repeats
 Usually 7-25 nucleotides connected to another, in up to
50 tandem repeats
 One person may them repeated 15x, person 2 is
repeated 35x at the same locus.
 Other terminology: STR: Short Tandem Repeats –
these are usually shorter sequences, repeated fewer
times.
 The STR sequences are of most important to use
in forensics, but there are other cases where
short repeated sequences on nucleotides can
have health effects as well.
 Huntington’s Disease: The CAG trinucleotide
repeat
 Normally: repeated 10-35 times within a gene
 Huntington’s: repeated 36 – 120+ times
 Because this region of the gene is excessively long, the
protein huntingtin is abnormally long as well. The
protein is cut into smaller a fragments by the body
which then accumulate around neurons and prevent
normal function of the nerve system.
 In forensics, we look at the variability in the
number and size of these STR’s because no two
people have exactly the same DNA codes in
these “nonsense” regions. (identical twins are
an exception)
 These differences are called polymorphisms –
and we use them to discriminate one person
from another.
Each chromosome has a different number
of the STR repeat – that have been
inherited from our parents.
We consider these alleles – just as we
would two copies of a gene on different
chromosomes.
 Example:
 Dad has the GGCT sequence repeated 3x on on
chromosome and 2x on the other. He is “heterozygous” for
this repeat because he has two different alleles. Referred
to as a (3,2)
 Mom has 4x and 5x repeats – also heterozygous (4,5)
 Offspring can be any of the four possible combinations:
3,4 .. 3,5 .. 2,4.. 2,5
Forensic
Applications
 http://
 There are two primary methods for selecting the
hypervariable regions of DNA for analysis.
 In both methods, the sequence is not actually read.
 These methods give us the ability to confidently rule out
possible suspects who do not match, and tell us who
cannot rule out.
 Restriction Fragment Length Polymorphism: RFLP
 Polymerase Chain Reaction: PCR
 Modern DNA analysis relies more so on PCR coupled with
other tests because PCR is capable of using much
smaller samples, badly damaged or degraded DNA. This
is useful for forensics because evidence is often sparse
and damaged.
 RFLP was one of the earlier DNA techniques used in
forensic analysis. It is much more direct, but requires a
large sample of DNA and often looks at much larger
sequences (VNTR) rather than STR.
 RFLP uses enzymes to cut the DNA into different sized
segments at specific sequences.
 The different sized pieces are then arranged by size and
this creates a pattern or a fingerprint for a person and a
particular sequence.
 These enzymes are called restriction endonuclease
enzymes and they recognize a specific nucleotide
sequence.
 Example: Hae III – “GGCC” – cuts between the G and C
 APA 1 – “GGGCCC” – cuts between the 1st and 2nd C
nucleotides
 More and more of these enzymes have been created as
their use in forensics has dramatically increased.
 Example: A restriction enzyme recognizes ACCT and cuts between the
two “C”’s . This cut is made every single time ACCT is recognized. This
results in many different sized fragments, depending on how many of
these ACCT a person has.
 Chromosome A – may cut it 5 times, each piece a different length of
base pairs (BP). Chromosome A’ – may cut 4 times.
 When run on a gel, the different sized pieces move through the gel
differently, depending on their size and charge.
 In a population, the number of times the ACCT sequence
is repeated can vary dramatically from person to person.
 This means that the size of the fragments produced from
this restriction enzyme is also highly variable.
 Databases have been created to show the frequency of
different fragment sizes, in order to provide statistical
support to the scientific evidence.
Population Frequency
 Population frequency is how common a particular DNA fragment size
is observed.
 Example: The frequency for on fragment size is 1 in 4 or 0.25 for
each chromosome.
 There is a 0.252 or 1/16 chance that someone else in the population
would match the fragment sizes on both chromosomes.
 As you increase the amount of bands (fragment sizes) that you
match, the chance of an accidental match decrease.
 Matching 10 bands would result in a 0.2510 chance – or 1 in
1,048,576. Matching 16 bands results in 1 in 4.3 trillion. This
statically analysis provides jurors, law enforcement with potentially
infallible evidence.
RFLP and Gel Electrophoresis
 The fragments move through the agarose gel at different
rates because of their size and charge.
 DNA is negatively charged because of the phosphate group.
When DNA is loaded into the wells, the wells are placed
near the negative electrode of the chamber so the repulsion
drives the DNA away.
 Friction prevents larger fragments from getting very far
away, while smaller pieces can easily move through the
porous material.
 In order to visualize the fragments, the gel must be
processed with procedures known as blotting and
hybridization.
 A nylon sheet or membrane that is positively charged is
layered and pressed upon the gel, and the DNA moves
from the gel to the membrane in the exact same
positions.
 DNA primers which are complimentary to the particular
sequence are applied, which allows them to bind to each
of the fragments in a different place.
 The primers also contain a radioactive or fluorescent tag.
 After hybridization, the membrane is placed next to
photographic film or UV light and the location of each
fragment is seen.
 With radioactive markers, the photographic film is
exposed and can then be developed.
 These are called autoradiographs because essentially the
DNA took “pictures” of themselves.
How is this used in forensics?
 Analysts will run the crime scene DNA evidence
alongside several suspects, all have been treating with
same restriction enzyme.
 By matching up DNA probes you can easily tell who you
can eliminate and which suspects you may need to
investigate further. You cannot use just one region to
convict – it takes many probes to prove beyond a
shadow of a doubt.
 Also used in inheritance/ paternity.
Polymerase Chain Reaction
 Due to the nature of DNA evidence at crime scene,
forensic analysts were often not able to conduct proper
RFLP testing.
 PCR was developed to take extremely small amounts of
DNA and copy selected fragments millions of times so
that there is enough to do an accurate analysis.
 It is considered an amplification process because many
copies are produced at a time. It has no direct forensic
application*
 PCR imitates the natural process of DNA replication with
a few slight modifications for our needs. There are 3
basic steps:
 1. Denaturation
 2. Hybridization
 3. Extension/ Ligation
 These steps all together are considered a cycle, and the
sequence of steps is regulated by adjusting the
temperature. PCR machines are often called thermocyclers.
 Denaturation:
 The double strands must be unraveled and separated
into single strands, to serve as a template.
 The hydrogen bonds are much weaker than the covalent
bonds holding the nucleotides together, so the DNA is
heated up to temperature of approximately 94oC.
 This breaks the hydrogen bonds, but leaves the covalent
bonds in tact, and separates the DNA into 2 templates.
 Hybridization/ Annealing
 The temperatures is lowered to 60oC, and the primers
are added. The temperature dictates how well the
primers anneal to the specific sequence.
 Primers mark the regions of DNA to be duplicated.
 They bind “anneal” to the beginning of the sequence of on
one strand of the DNA.
 They bind or flank the end of the sequence of the other
strand of DNA
 Remember, the 3’ and 5’ ends of DNA. This is why the
primers bind to the beginning on the 5’ strand and the end
on the 3’ strand. This is because Taq DNA Polymerase only
synthesize DNA in one direction: 5’  3’
Extension
 The temperature is raised again, to the optimal temperature for the
enzyme taq polymerase – about 75 – 80oC.
 Taq polymerase is used because it is able to withstand changes in
temperature – “thermostable”
 Taq DNA polymerase is able to find these primers and fill in the rest
of the complimentary strand in a 5’ to 3’ direction. This is the step
known as extension.
 At this point, there are the two original templates and
the two new complimentary strands.
 The temperature is raised again to break any hydrogen
bonds and so that each of the four pieces can be used as
template again. However, the size of the “new”
templates is different from the original since only a
certain segment was duplicated.
 This results in eventually many copies of the segment of
interest as they can constantly be used as templates.
PCR Animations
 http://www.dnalc.org/resources/animations/pcr.html
 http://www.maxanim.com/genetics/PCR/pcr.swf
 http://www.invitrogen.com/site/us/en/home/Productsand-Services/Applications/PCR/elevate-pcr-research/pcrvideo-library/pcr-animation.html
 http://learn.genetics.utah.edu/content/labs/pcr/
STR Analysis
 Short Tandem Repeats are much smaller in
both size and the number of repeats than the
VNTR’s. Sometimes referred to as
microsatellites.
 Their smaller size is useful particularly when
the DNA is badly damaged or degraded. Easy
to amplify through PCR.
 The number of STR’s you have are still
inherited from each parent, and the different
number of STR’s you have at a particular
locus is still called an allele.
 Combined DNA Index System (CODIS)
 FBI only uses STR loci with 4 base pairs
although in general they can range from
2-6 base pairs.
 FBI uses 13 loci for matching purposes
 Interpol has established 10 -12 (April
2009) STR loci for European populations
 9 STR loci have been standardized for
Indian populations
 There are many more than just 13 STR loci
that exist within our DNA, however we do not
have population frequency for them, primers
have not been developed to allow for their
amplification.
 As more of these STR loci have been studied
and tweaked for use in forensic analysis, there
has been discussion about adopting more STR
loci for two reasons:
 Try to reduce the chance of a random match
 International compatibility of DNA databases
 Out of the 13 STR loci the FBI uses, only 8 of them
overlap with loci used in Europe.
STR DNA Fingerprints
 STR analysis uses PCR amplification: Results in large
number of copies of the STR loci that we are looking at.
 Different from RFLP which uses restriction
endonucleases to “cut” the DNA at certain segments.
RFLP is a more direct approach, but requires a lot of
DNA and is very time consuming.
 Because STR’s are smaller and do not overlap (we will
discuss details shortly), we are able to perform PCR and
amplify up to 9 different STR loci in a single reaction.
STR DNA Fingerprints
 The PCR process is nearly identical to the PCR
discussed earlier: Denaturation, Annealing,
Elongation.
 However, when the oligonucleotides (PRIMERS) are
added to the reaction mixture, a small change has
been made.
 The primers are tagged with a molecule that will
fluoresce when exposed to a certain wavelength of
light. This allows us (gel electrophoresis) or
machines (capillary electrophoresis) to visualize the
different fragments.
Capillary Electrophoresis
 Technique very similar to gel electrophoresis, relies on a
detector and computer software to quantify what we
“visualize”
 Rather than use a flat gel plate, the DNA fragments that
were amplified in PCR move through a charged glass
capillary tube, amount the size of a human hair.
 The fragments are carried by a solvent, with the smaller
pieces reaching the end of the tube first.
 However, rather than letting the solvents run and then
comparing the bands, we analyze the fragments as they
pass by to get a DNA fingerprint.
With the development of STR analysis,
there are two types of data usually
associated with this testing:
Gel Electrophoresis: Bands to show relative size
Capillary Electrophoresis: Peaks to show
relative size
Advantages are:
 smaller quantities of sample necessary and can be
retested easily.
 Data is collected and viewed real time on a computer.
 Bubbles and gel thickness doesn’t diminish
reproducibility.
 Less time required, fully automated, requires less
manual labor
Capillary Electrophoresis:
 Animation:
 http://www.wiley.com/college/pratt/0471393878/studen
t/animations/dna_sequencing/index.html
 The DNA fingerprint that is created from capillary
electrophoresis doesn’t rely on bands, it relies on
peaks.
 As the fragments pass by the laser and are
analyzed by the detector (photocell). The laser
excites the dye molecules, which cause them to
fluoresce.
 The photocell and computer can detect the
intensity, wavelength and color of the fluorescence
– which creates peaks as each band passes
through.
Results from Capillary Electrophoresis:
Comparing RFLP, Gel
Electrophoresis and Capillary
Electrophoresis:
Autoradiograph:
Comparing RFLP, Gel
Electrophoresis and Capillary
Electrophoresis:
DNA Fingerprint
Bands
Comparing RFLP, Gel
Electrophoresis and Capillary
Electrophoresis:
Peaks:
STR Analysis Continued…
 Locus D13S317 is a STR loci that the FBI
uses – there are 9 possibilities of how
many repeats exist:
5,7,8,9,10,11,12,13,14 and 15 repeats of
the TATC sequence.
 The population frequencies of these alleles
have been determined which will allow us
to statistical analysis to determine how
reliable a “match” is for this loci.
Because of difference between populations, the
frequency differs from country to country:
The 5 repeat: unknown in Australia, Germany, US
however occurs in 5.3% of Chinese population.
15 repeat: 3.3% of American, does not appear in
Germany or China, 1.6% of Australian.
 To determine the odds of a random match for a
particular locus –
 You multiply the probabilities of each allele together.
 For example: in the United States that chance of
having a 9 repeat and a 11 repeat:
 The probability of 9 repeats: 0.0762
 11 repeat: 0.3377
 The resulting probability: 0.0257 or 2.57%
 That means that 2.57% of population in the United
States shares both the 9 and the 11 repeat.
 As you do this for 13 loci, the odds that a
random match occurs within the
population diminishes greatly.
 In order to look at many STR’s at the
same time, a technique called multiplexing
was developed.
 Multiplexing uses different fluorescent
tags and different primers that anneal to
specific loci and can all be amplified at the
same time.
Multiplexing
When multiplexing, there are several things to consider.
You can’t select loci randomly.
Locus size and overlap
Number of alleles
Thermal cycling requirements
Primers
Dyes
Locus Size and Overlap:
 Most STR of interest are between 75-400 base pairs long.
When selecting which loci to amplify at the same time, they
have competing sizes – some of which may be similar.
 This will blur the results (peaks) and you will not be able to
correlate peaks with a particular loci.
 Studies have been done to find that allele size region of many
of the forensically important STR loci.
 A multiplex works best when the smallest allele for one loci is
larger than the largest allele of the next loci if they are tagged
with the same dye.
 Source: www.nftsc.org/pdi/subject04/pdi_s04_m03_01_a.htm
 Because a multiplex amplifies many different STR: the
sizes of all possible alleles plays an important role in
which ones we can amplify simultaneously.
 If we used a different fluorescent dye for each STR,
then if we had size overlap it wouldn’t matter, because
the peaks would be different colors.
 However, there is a limit to how many fluorescent dyes
we have available, which means some STR’s that are
run at the same time might have the same fluorescent
tag. To tell these apart, we have to take into account
overlapping allele sizes.
 Ideally, running STR’s with the same dye – you want
the largest size of the 1st allele is smaller than the
smallest allele of the 2nd STR allele.
Number of alleles…
 For a single locus STR analysis – the more alleles
(number of repeats) – the more variation. This
gives it more discriminating power to discern
between two individuals.
 However, when running a multiplex with STR loci
that have many alleles, there is a greater chance
that they will overlap with the loci that comes
before and after.
 Other considerations:
 Primers – have temperatures that they work
best at – optimum annealing temperatures. You
have to select loci that have primers that are
flexible in their performance at different
temperatures.
 Primers – have to select primers that will not
interact with each other.
 Selecting dyes that give you individual peaks in
a variety of colors.
 Interpreting STR Analysis Data:
 The horizontal axis is the number of nucleotides,
the vertical axis is gives an indication of relative
peak height.
 Because we each have two alleles for an STR loci
– there can be two peaks arising due to the two
different sized fragments – but only if the person
is heterozygous.
 If the person is homozygous, you will see only
one peak for an STR loci, you will only see one
peak, but it will be much taller due to more DNA
that is that particular size.
Additional types of DNA profiling
 The issue of badly degraded DNA samples continues to challenge
forensic scientists to develop ways of using smaller pieces of DNA to
identify matches.
 This leads to Mini-STR and “SniPs” or SNP Profiling
 In Mini-STR – the primers are moved much closer to the actual STR loci
– means the pieces that are being amplified are much smaller – less
wiggle room.
 SNP – single nucleotide polymorphism
 One base pair is different than majority of population
 Because changes are quite common – you need many of these SNP to create a
match – takes a lot of time. May replace STR in the future.
SNP’s
 Advantages: work well on degraded DNA
because you are amplifying smaller
regions of DNA. Lower mutation rate over
STR’s (better inheritance reliability)
 Disadvantages: Need to look at 40-60 loci
to get a match (13-15 STR). Results are
hard to interpret.
 Could be used to paternity/ancestry
studies. More work to be done before
replaces STR in forensics.
Mitochondrial DNA
 Small circular loop of DNA found in Mitochondria,
only inherited from mother.
 Why?
 Egg cells have 100,000+ mitochondria. Sperm cells only a
couple of hundred located in the flagellum.
 During fertilization, the flagellum never penetrates the egg.
So Dad’s mitochondria never are introduced into the zygote.
 Can only use to identify same matrilineal descent, Can’t use
MtDNA to distinguish between siblings.
 mtDNA is used because each cell have tens of thousands o
mitochondria with the same copy of mtDNA. We only have
one nuclear DNA per cell = Lots more mitochondria, more
likely to have some mtDNA left over in a badly damaged
sample.
mtDNA
 mtDNA is mostly composed of coding
regions – very little junk DNA.
 Approx 16,569 nucleotides and only 1200
are non coding and referred to as
the”control region”
 Within the control region, there are
differences in the sequence – and can be
used to compare between two sample to
try to find a matrilineal relationship.
Y chromosome DNA
 Inherited from fathers – can be used to link males and
other paternal relatives.
 Often used to distinguish between males in sexual
assault cases. During PCR, the primers used to amplify
regions on Y chromosome ignore any female DNA.
 Only amplify DNA from male, it is a way of separating
out and analyzing the male DNA without having to
physically separate the mixture.
 Approx 60% of Y chromosome is repetive sequences,
can be used to compare and look for matches.
Science in the Courtroom…
Evolution of DNA Evidence
Tommie Lee Andrews: 1st US Conviction
using DNA evidence
 Background Information: May 1986: Man entered
Nancy Hodge’s apartment, raped her at knifepoint and
stole purse. Continued pattern for 6 months, raping 23
women and taking items from each rape.
 Fingerprint taken from windows of victim apartment,
match his when he was brought in as a “prowler”.
 Police noticed a pattern between 23 rapes, but only one
woman got a brief glimpse of him, and could only prove
his blood type matched semen. Not enough to prove
serial rapist.
 Tommy Lee Andrews – charged with 6 separate
counts of extremely violent rape. Being prosecuted
by Tim Berry.
 Tim Berry was contacted by Jeffrey L. Ashton, the
assistant state attorney for state of Florida. Ashton
saw an ad from Lifecodes based in Valhalla, NY for
paternity testing using DNA.
 Sent semen samples from 6 victims and Andrews to
LifeCodes and they reported that their was a match
in 2 of the 6 victims. Prosecution prepared for two
trials.
After a Frye hearing, judge allowed the DNA evidence. During 1st
trial, prosecution exaggerated the odds of DNA testing, hung jury,
mistrial.
2nd trial: Dr. Michael Baird from Lifecodes testified that semen
sample from victim matched blood sample from accused. Also
provided statistical evidence: The chance that the DNA could
belong to someone else other than Andrews was 1 in
839,914,540.
Lab tech Alan Guisti also testified regarding protocols, samples,
procedures followed. Dr. David Housman from MIT also testified
to the reliability and accuracy of DNA identity testing.
Andrews convicted and sentenced to 22 years. Was then retried
for 1st charge and was also convicted. Sentenced to 115 years for
serial rape.
 Recent developments in Andrews case:
 Judge reduced the sentence from 115 years to 62 years and then
again for good behavior.
 In Oct 2012, Tommy Lee Andrews is acting as his own lawyer in a
Jimmy Ryce hearing. Would like to be released from prison and
avoid going to sex offender treatment.
 State psychologists have testified that he is still a risk, because he
has never admitted guilt, sought treatment while in prison, admitted
mental problems, refused to be examined by state doctors.

Summary
 The Andrews case was the 1st case to use DNA to convict.
 The judge allowed the DNA evidence to be admissible under
the Frye standard and commented on the impressive
credentials of the expert testimonies.
 Used RFLP and gel electrophoresis, DNA evidence
considered highly accurate. Andrew’s defense attorney had
nothing to counteract the DNA evidence.
 DNA evidence was unchallenged, poorly understood but
passed Frye hearings. Beginning of a time when the
scientific and legal systems had to try to introduce new
technology and knowledge, in a way that was
understandable, credible and trustworthy.
People vs. Castro
 People vs. Castro – 1989. 1st documented case where
DNA evidene was challenged.
 Longest pre-trial Frye hearing in history of Supreme
Court.
 Background Information: 1987 – the Bronx – Joseph
Castro charged with murdering his neighbor and her two
year old daughter. The forensic evidence in question
was a blood stain on his watch.
 Two defense lawyers: Barry Scheck and Peter Neufeld – 1st
challenge to DNA evidence.
 Blood samples from watch and victims were sent to
Lifecodes. A match between blood from the victim and blood
on the watch was reported. Chance of match being random
was 1 in 189,200,000.
 Prosecution wanted this entered as evidence, defense
challenged.
 1st challenge: Lifecodes was private, could not be held
accountable for protocol. Lifecode counteracted that their
practices were accepted by scientific community, but were also
trade secrets.
 Scheck and Neufeld attend conference by Dr. Baird – he
admitted to declaring a match, even though some bands were
misaligned on the blot (autorad). Dr. Eric Lander also attended
and was disturbed by the amount of “fudging.”
 Defense lawyers asked Lander to look at Lifecodes results for
the Castro case.
“ As Neufeld tells the story, Lander responded, ‘Let
me show you how we do things in science’. Lander
then called over several colleagues and slapped the
autorad up against the window and said, ‘Match or
no match?’
‘Garbage,’ one man reponded.
‘ Do it again,’ said another.
‘Garbage,’ said a third. “
Source: http://web.viu.ca/derksenl/Publications/Derksen%20Chapter%203%20April%202006.pdf
 Defense subpoenas Lifecodes data
 For one locus: Lifecodes reported a band to be
10.25kb long, for both the watch and the victim.
 Records indicate that the watch stain was 10.16kb
long, and victims blood was 10.35kb. Lifecodes
averaged them and reported them to be the same
size.
 Additionally, 10.16 and 10.35 are outside the 3 std
dev limit that Lifecodes had reported was it’s
standard procedure.
 Out of the 6 matching bands that Lifecodes
reported, two of them fell outside of the 3
standard deviations. There was also a band
present on the victim, that was missing on the
watch and Lifecodes omitted it from the report.
 Dr. Baird for the prosecution testified that they
were non-human DNA contaminants. Dr. Lander
for the defense eventually testified and provided
reasonable doubt in Dr. Baird’s explanation.
 After 15 weeks of pre-trial hearings, the judge
ruled that although DNA typing was accepted in
the scientific community, Lifecodes had failed to
perform scientifically acceptable tests and had
failed to obtain reliable results.
 Follow up: Castro’s case never went to trial. He pled guilty for
a lesser sentence after the DNA evidence was excluded.
 The defense lawyers exposed the variability in DNA testing,
showing that the reliability depends greatly on adhering to
standards and practices.
 Scheck and Neufeld became members of a DNA task force ,
who helped defense lawyers attack DNA testing. They sought
out scientists who were apprehensive about DNA typing.
 DNA evidence that was previously uncontested, was being
denounced from 1987-1991. Known as the “War on DNA.”
Results from Castro:
 DNA Identification theory and practice are generally accepted
among scientific community
 DNA forensic identification techniques are generally accepted by
the scientific community.
 Pretrial hearings are required to determine whether the DNA lab
methodology was aligned with scientific standards.
 Also supports that DNA is more admissible when being used for
exclusion rather than inclusion.
 There are two exemplar cases that highlight the use of nonhuman DNA to identify criminals
 1994: Snowball the Cat
 1995: Palo Verde Tree
Snowball the Cat
 Background: Prince Edward Island, 1994.
 Shirley Duguay disappeared and her body was
found in a shallow grave several months later.
Investigators suspected her common law husband,
Douglas Beamish who lived nearby.
 The primary piece of evidence that they discovered
was a plastic bag that contained a leather jacket,
with blood stains.
 Snowball the Cat:
 The blood on the jacket was determined to be from the
victim, but they lacked evidence to link Beamish to the crime.
 The jacket also contained 27 strands of white hair, which was
determined to have came from a cat. Investigators
remembered a cat that lived in Beamish’s parent’s home. But
in order to prove the think, they had to prove that the hair
from the jacket, came from Snowball.
 They isolated the DNA and performed a DNA fingerprint on
the cat hair from the jacket, and from a blood sample
collected from Snowball. Beamish was convicted of murder.
 Investigators also tested 20 other cats from
Prince Edward Island to get an idea of the
genetic variability among the cat population.
 First case to allow animal DNA-typing
evidence. There is now research being
conducted to create a DNA profile database
for different cat breeds.
1995: The Bogan Case: “The Tree
that was a Murder Witness”
 Nude body of a woman discovered in the desert
near a cluster of palo verde trees. A man in the
area said that witnessed a truck leaving the area
at approx 1:30am.
 During the investigation: police found a pager
belonging to Earl Bogan. Used primarily by his
son Mark, who drove a white pickup truck.
 The truck was then combed over by the CSI
team and two palo verde seed pods were found
in the truck.
 The original scene was revisited and there was
a fresh scratch on one of the palo verde trees
(PV-30).
 Prosecutors reached out to a molecular
genetics professor at University of Arizona, who
compared the DNA from the truck seed pods to
the seed pods from PV 30 and concluded there
was a match.
 Mark Bogan was convicted of murder and then
appealed the decision.
 During the appeal, Bogan fought that the
professors testimony should be excluded. Dr.
Helentjaris had testified that the odds were 1 in a
million which the judge ruled inadmissible.
 Dr. Helentjaris also said “he felt quite confident in
concluding that these two samples… most likely
did come from PV-30” and he was “quite
comfortable” stating that the PV30 DNA was
distinguishable from the DNA of other trees.
 However, he didn’t testify with his actual
calculations, which provided an opportunity for
Bogan and the defense to try to exploit. However,
the court of appeals held up the conviction.
MtDNA evidence
mtDNA has been increasingly used to re-exam cold
cases, most often those dealing with hair evidence.
Hairs as small as 2mm and 40 years old have been
successfully tested and result in a DNA profile, cases
all the way back to the 1970’s can be re-opened.
Hair microscopy and mtDNA testing usually used
together:
- When hair is analyzed for mtDNA – 94.7%
conclusive results
- When hair is analyzed by microscopy – 58.2%
conclusive results
Case #1: 1999 - Terrorist Storage Locker – 1970’s
Armenian nationalist rent a storage locker for guns
and explosives. When rent was not paid in 1996 –
the locker was opened and then investigated.
 Several hair fragments were collected and
eventually tied back to the leader of a terrorist
group: Mourad Topalian.
 This used mtDNA analysis similar to what would
be done on ancient remains, because the DNA
was damaged and there was very little of it
remaining. Topalian was convicted, sentenced to
37 months in prison.
 Case #2: William Gregory Exoneration
 William Gregory was identified in a lineup as the man
who raped a woman in his apartment complex.
 The only evidence was 6 head hairs from the
pantyhose of the victim, used as a mask. The hairs
were analyzed using hair microscopy. The microscopist
concluded that the hairs could have come from
Gregory. He was eventually convicted.
 In 2000, mtDNA analysis was done – the six hairs had
the same DNA profile, but it did not match Gregory or
the victim. Gregory was released from prison and the
case remains open.
Murder of Laci Peterson