Investigating CSI – Background material Table of Contents I ...

Investigating CSI – Background material
Table of Contents
I. Overview of Forensic Science and DNA Evidence
A. Marie Campbell lecture (pgs. 2-5)
B. Overview of Forensic Identification (pgs. 6-9)
C. Overview of DNA Evidence (pgs. 10-19)
II. Tools and Techniques for DNA Forensic Science
A. Detection of Body Fluids
i. Ultraviolet Light Illumination and Microscopy (pg. 21)
ii. Acid Phosphatase Test (pg. 22)
iii. p30 Antigen Test (pg. 23)
iv. The Kastle-Meyer Test (pg. 24-29)
v. Luminol (pgs. 30-31)
vi. Amylase test (pgs. 32-33)
B. Isolation of DNA
i. Differential Lysis (pg. 34)
ii. DNA Extraction (pgs. 35-36)
C. Quantification of DNA
i. Southern Blots (pgs. 37-39)
ii. Real-time PCR (pgs. 40-43)
D. Creating a DNA profile
i. Restriction Fragment Length Polymorphism (pgs. 44-50)
ii. Short Tandem Repeat (pgs. 51-55)
E. Other Issues
i. Mitochondrial DNA (pgs. 56-57)
ii. Y-Chromosome Analysis (pg. 58)
III. Interesting Cases using DNA evidence
A. Snowball the Cat (Pgs. 59-61)
B. OJ Simpson Murder Trail (pgs. 62-64)
C. Czar Nicholas II (pgs. 65-67)
D. John Doe case (pgs. 68-71)

I. OVERVIEW OF FORENSIC SCIENCE DNA EVIDENCE
The work of the forensic scientist: IBMS West of Scotland Branch Meeting
A well-attended IBMS West of Scotland branch meeting in early November
saw Marie Campbell from Strathclyde Police forensic science department
give a fascinating overview of the work of a forensic scientist.
Her first point was to explain the difference between a forensic scientist and a
forensic pathologist - the latter deals with autopsies while a forensic scientist
deals with the scene of crime, evidence investigation and DNA testing.
Forensic science divides into forensic chemistry and forensic biology. Forensic
chemistry deals with drug testing, marks and unique striations on tools used in
crimes such as burglary, footwear marks, fire scenes, firearms and shooting
incidents, forged documents and handwriting, threatening letters or money.
Drug testing is the perhaps the busiest role with thousands of cases processed
every year. Fire debris is sifted through to find small pieces of evidence that
would suggest a deliberate cause. The way a fire 'naturally' takes hold in a room
indicates whether it was started deliberately but evidence can still be hard to find
when it is covered by debris or a collapsed ceiling.
Physical chemistry looks at footwear comparison and gelatine lifting off a
suspect's shoes. Footwear comparison involves the electrostatic lifting of
footwear if a burglar has stood, for example, on a newspaper. The footprint can't
be seen but dust lifted off the paper to see the print on foil will show the unique
footwear marks that are made by an individual because of the way he or she
'treads' and wears the shoes.
Forensic chemistry also looks at paint comparisons - a trace of paint in a hit and
run accident can link back to a vehicle. Marie illustrated the point in a local case
where a kidnapper hit a tree before making his escape. Laboratory analysis of
the paint left on the tree was able to confirm manufacture of the car and the year
it was made allowing the police to quickly identify local suspects and find the
kidnapper.
Chemical etching is another technique employed to uncovered erased serial
marks. The plate can be dipped sulphuric acid to find out the original marks.
Firearms Discharge Residue analysis is another area of investigation. Residue
can be found on hands, clothing and in vehicles - part of the evidence used to
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convict the killer of Gill Dando was one particle of firearm residue. Forensic
chemists can classify the make of the firearm and link the bullet to the gun.
UV traps are used to mark money in, for example, a company office, which is
experiencing an outbreak of theft. Chemicals on the marked money is made with
a special, secret combination known only to the police. A swab from the
suspect's hand will confirm the combination. The combination of chemicals
eliminates contamination from possible sources other than the marked money -
such as, strangely enough, oranges.
Forged signatures, photocopies, handwriting, document alterations are also
studied using ESDA examinations for different indentations of writing.
A vast amount of laboratory testing is done at the Strathclyde forensic biology
department to answer questions about body fluids, whether they are human,
where they come from and whose is it. A KM test that reacts with haemoglobin in
blood confirms that a substance is indeed blood. Blood pattern analysis will
answer further questions and give clues to building a picture of what has actually
happened in an incident.
A suspect has blood on his or her clothes but how the blood is on the clothes
offers clues to the participation in the incident. Blood stains indicate direct blood
touching which could mean the suspect has cradled the victim or tried to help.
Blood spots on clothes tell a different story as spots can only be caused by
violent impact, which would disprove a suspect's claim that he was trying to help
the victim.
Marie Campbell showed examples of this with photos of a pair of trousers
showing the blood spot splatter caused by kicking the victim and a jacket with
contact bloodstains of a passer-by who has stopped to help the victim. Analysis
also looks at the lines of blood flying from the weapon as it is brought up and
then down on a victim.
Blood analysis determines species identification and blood grouping using two
systems - DNA profiling or PCR. The laboratory will analyse 10 areas of DNA
and conduct sex test that ensure the chances of a false identification are one in a
billion.
The advantages of DNA testing are that very small sources can be used and
information can be extracted from very old stains. Testing can also be done on
different sources such as hair although it has to have root material for successful
testing. DNA testing can also be done on semen, body tissue such as deep
muscle tissue within a decomposed torso, saliva (cigarette ends, bottles, gags,
stamps) and skin cells found in faeces, urine, sweat and dandruff. Urine itself
does not contain DNA - it is the cells being passed through urine that carries
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DNA information. DNA testing can also be done on weapons - knifes collect skin
cells that can be profiled.
Sifting through scenes of crime for possible sources of evidence requires lateral
thinking and nothing can be discarded in case it develops significance later in a
police investigation. DNA testing now uses the quicker method of profile numbers
rather than 'barcodes'.
In sexual offences acid phosphate tests are used to identify semen. However, the
department also investigates the damage of underwear to determine whether the
damage is recent or self-inflicted. The ends of fibres can tell whether the
underwear is torn, cut or just very old. This type of investigation is obviously
important in rape cases.
Analysis of hairs and clothes fibres is another way of gathering evidence
although the success of DNA has meant that hair analysis is now rarely used.
Fibres can provide excellent proof of recent contact and can be used to back up
DNA evidence. Lateral thinking is needed in looking for samples - for example
how someone would have escaped from a crime and thus possibly left samples
while climbing a wall.
Forensic biologists also attend scenes of crime which can cover very large areas
- especially if streets have to be closed down. Forensic biologist can find
themselves in difficult working conditions - freezing cold fields in the middle of the
night or derelict buildings. Marie pointed out that TV programmes on forensic
examinations where sharp suited detectives wandered through the crime scene
instantly deciphering clues were far removed from reality. Scenes of crime were
now overseen by managers who ensure that the sites are secure to preserve
evidnece. Forensic scientists dress in all over white suits to avoid not only
contaminating evidence but also the evidence contaminating workers.
The National DNA Database was established in 1995 and holds DNA profiles
from mouth swabs (Criminal Justice samples - CJ)) of convicted criminals and
records of outstanding crimes. The database is run by the Forensic Science
Service in Birmingham although there are also local facilities at Dundee. The
database currently holds around two million CJ samples and 160,000 crime
scene profiles. Obviously the use of the database to solve crime can only
succeed if a suspect has committed a previous crime but since 1995 a large
amount of violent crime has been solved by running crime scene profiles against
CJ samples - including 1,000 murders and 1,800 rapes. Cases from the past
have been solved as the same person commits new crimes or new CJs are
logged on the database.
The lecture was followed by a question and answer session that included a
number of points including the necessary qualifications to enter a career as a
forensic scientist, recruitment and retention and quality audits.
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A forensic scientists needs a honours degree in biology and chemistry and a
masters degree in forensic science to enter the profession. An forensic assistant
will need an aapropriate HND or more.
The high media profile of the work of a forensic scientist means that retention is
more of a problem than recruitment. Marie Campbell believes that many come
into the job believing it to be the kind of glamorous career profiled in television
crime dramas and can be quickly disillusioned by the reality. While there was
immense personal satisfaction it can be a hard job that should be considered as
a vocation that someone would really want to enter as a career. Marie highlighted
the obvious unpleasantness of attending a particularly nasty scene of crime or
accident. There was also the added stress of being cross-examined in court even
though that was something a scientist quickly becomes accustomed to.
Was television giving the game away on how forensics can solve crime? Most
crimes are committed out of rage, passion or are alcohol fuelled - whatever the
motives few crimes are committed in such a rational, calm and 'intelligent'
manner that would enable criminal to think and cover their tracks. Many suspects
also tripped themselves up by insisting on versions of stories that conflicted with
the facts uncovered by forensic investigation.
The forensic laboratory with Strathclyde police is externally accredited and also
undergoes internal audit once every two to three months. Chain of custody
procedures with regard to transferring evidence from the laboratory to court are
also rigorously
The lecture was a fascinating insight into the work of a forensic scientist with
illuminating examples of local and national cases where forensic investigation
had helped to solve crime.
Source: http://www.ibms.org/index.cfm?method=science.general_science&subpage=general_forensic_scientists
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How does forensic identification work?
Any type of organism can be identified by examination of DNA sequences unique
to that species. Identifying individuals within a species is less precise at this time,
although when DNA sequencing technologies progress farther, direct comparison
of very large DNA segments, and possibly even whole genomes, will become
feasible and practical and will allow precise individual identification.
To identify individuals, forensic scientists scan 13 DNA regions that vary from
person to person and use the data to create a DNA profile of that individual
(sometimes called a DNA fingerprint). There is an extremely small chance that
another person has the same DNA profile for a particular set of regions.
Some Examples of DNA Uses for Forensic Identification
• Identify potential suspects whose DNA may match evidence left at crime
scenes
• Exonerate persons wrongly accused of crimes
• Identify crime and catastrophe victims
• Establish paternity and other family relationships
• Identify endangered and protected species as an aid to wildlife officials
(could be used for prosecuting poachers)
• Detect bacteria and other organisms that may pollute air, water, soil, and
food
• Match organ donors with recipients in transplant programs
• Determine pedigree for seed or livestock breeds
• Authenticate consumables such as caviar and wine
Is DNA effective in identifying persons?
[answer provided by Daniel Drell of the U.S. DOE Human Genome Program]
DNA identification can be quite effective if used intelligently. Portions of the DNA
sequence that vary the most among humans must be used; also, portions must
be large enough to overcome the fact that human mating is not absolutely
random.
Consider the scenario of a crime scene investigation . . .
Assume that type O blood is found at the crime scene. Type O occurs in about
45% of Americans. If investigators type only for ABO, then finding that the
"suspect" in a crime is type O really doesn't reveal very much.
If, in addition to being type O, the suspect is a blond, and blond hair is found at
the crime scene, then you now have two bits of evidence to suggest who really
did it. However, there are a lot of Type O blonds out there.
If you find that the crime scene has footprints from a pair of Nike Air Jordans
(with a distinctive tread design) and the suspect, in addition to being type O and
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lond, is also wearing Air Jordans with the same tread design, then you are
much closer to linking the suspect with the crime scene.
In this way, by accumulating bits of linking evidence in a chain, where each bit by
itself isn't very strong but the set of all of them together is very strong, you can
argue that your suspect really is the right person.
With DNA, the same kind of thinking is used; you can look for matches (based on
sequence or on numbers of small repeating units of DNA sequence) at a number
of different locations on the person's genome; one or two (even three) aren't
enough to be confident that the suspect is the right one, but four (sometimes five)
are used and a match at all five is rare enough that you (or a prosecutor or a jury)
can be very confident ("beyond a reasonable doubt") that the right person is
accused.
How is DNA typing done?
Only one-tenth of a single percent of DNA (about 3 million bases) differs from
one person to the next. Scientists can use these variable regions to generate a
DNA profile of an individual, using samples from blood, bone, hair, and other
body tissues and products.
In criminal cases, this generally involves obtaining samples from crime-scene
evidence and a suspect, extracting the DNA, and analyzing it for the presence of
a set of specific DNA regions (markers).
Scientists find the markers in a DNA sample by designing small pieces of DNA
(probes) that will each seek out and bind to a complementary DNA sequence in
the sample. A series of probes bound to a DNA sample creates a distinctive
pattern for an individual. Forensic scientists compare these DNA profiles to
determine whether the suspect's sample matches the evidence sample. A marker
by itself usually is not unique to an individual; if, however, two DNA samples are
alike at four or five regions, odds are great that the samples are from the same
person.
If the sample profiles don't match, the person did not contribute the DNA at the
crime scene.
If the patterns match, the suspect may have contributed the evidence sample.
While there is a chance that someone else has the same DNA profile for a
particular probe set, the odds are exceedingly slim. The question is, How small
do the odds have to be when conviction of the guilty or acquittal of the innocent
lies in the balance? Many judges consider this a matter for a jury to take into
consideration along with other evidence in the case. Experts point out that using
DNA forensic technology is far superior to eyewitness accounts, where the odds
for correct identification are about 50:50.
The more probes used in DNA analysis, the greater the odds for a unique pattern
and against a coincidental match, but each additional probe adds greatly to the
time and expense of testing. Four to six probes are recommended. Testing with
several more probes will become routine, observed John Hicks (Alabama State
Department of Forensic Services). He predicted that, DNA chip technology (in
which thousands of short DNA sequences are embedded in a tiny chip) will
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enable much more rapid, inexpensive analysis using many more probes, and
raising the odds against coincidental matches.
What are some of the DNA technologies used in forensic investigations?
Restriction Fragment Length Polymorphism (RFLP)
RFLP is a technique for analyzing the variable lengths of DNA fragments that
result from digesting a DNA sample with a special kind of enzyme. This enzyme,
a restriction endonuclease, cuts DNA at a specific sequence pattern know as a
restriction endonuclease recognition site. The presence or absence of certain
recognition sites in a DNA sample generates variable lengths of DNA fragments,
which are separated using gel electrophoresis. They are then hybridized with
DNA probes that bind to a complementary DNA sequence in the sample.
RFLP is one of the original applications of DNA analysis to forensic investigation.
With the development of newer, more efficient DNA-analysis techniques, RFLP is
not used as much as it once was because it requires relatively large amounts of
DNA. In addition, samples degraded by environmental factors, such as dirt or
mold, do not work well with RFLP.
PCR Analysis
PCR (polymerase chain reaction) is used to make millions of exact copies of
DNA from a biological sample. DNA amplification with PCR allows DNA analysis
on biological samples as small as a few skin cells. With RFLP, DNA samples
would have to be about the size of a quarter. The ability of PCR to amplify such
tiny quantities of DNA enables even highly degraded samples to be analyzed.
Great care, however, must be taken to prevent contamination with other
biological materials during the identifying, collecting, and preserving of a sample.
STR Analysis
Short tandem repeat (STR) technology is used to evaluate specific regions (loci)
within nuclear DNA. Variability in STR regions can be used to distinguish one
DNA profile from another. The Federal Bureau of Investigation (FBI) uses a
standard set of 13 specific STR regions for CODIS. CODIS is a software program
that operates local, state, and national databases of DNA profiles from convicted
offenders, unsolved crime scene evidence, and missing persons. The odds that
two individuals will have the same 13-loci DNA profile is about one in one billion.
Mitochondrial DNA Analysis
Mitochondrial DNA analysis (mtDNA) can be used to examine the DNA from
samples that cannot be analyzed by RFLP or STR. Nuclear DNA must be
extracted from samples for use in RFLP, PCR, and STR; however, mtDNA
analysis uses DNA extracted from another cellular organelle called a
mitochondrion. While older biological samples that lack nucleated cellular
material, such as hair, bones, and teeth, cannot be analyzed with STR and
RFLP, they can be analyzed with mtDNA. In the investigation of cases that have
gone unsolved for many years, mtDNA is extremely valuable.
All mothers have the same mitochondrial DNA as their daughters. This is
because the mitochondria of each new embryo comes from the mother's egg cell.
The father's sperm contributes only nuclear DNA. Comparing the mtDNA profile
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of unidentified remains with the profile of a potential maternal relative can be an
important technique in missing person investigations.
Y-Chromosome Analysis
The Y chromosome is passed directly from father to son, so the analysis of
genetic markers on the Y chromosome is especially useful for tracing
relationships among males or for analyzing biological evidence involving multiple
male contributors.
Source: http://www.ornl.gov/sci/techresources/Human_Genome/elsi/forensics.shtml
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How DNA evidence works
The public has always been captivated by the drama that occurs in the
courtroom. There is even a whole channel, CourtTV, devoted to showing real
court cases as they wend their way through the legal system. TV shows and
movies depict passionate attorneys sparring verbally as they fight to convict or
acquit the accused. However, the most tense moments of a criminal trial are
likely those that go unseen: the jury deliberations.
After both sides present their evidence and argue their cases, a panel of jurors
must weigh what they have heard and decide whether or not the accused person
is guilty as charged. This can be difficult. The evidence presented is not always
clear-cut, and sometimes jurors must decide based on what a witness says they
saw or heard. Physical evidence can be limited to strands of hair or pieces of
fabric that the prosecution must somehow link conclusively to the defendant.
What if there was a way of tying a person to the scene of a crime beyond a
shadow of a doubt? Or, more importantly, what if you could rule out suspects and
prevent the wrong person from being locked up in jail? This dream is beginning
to be realized through the use of DNA evidence. In this article, we'll look at how
DNA "fingerprinting" works and find out what DNA evidence can be used for.
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Matching DNA
Proving that a suspect's DNA matches a sample left at the scene of a crime
requires two things:
• Creating a DNA profile using basic molecular biology protocols
• Crunching numbers and applying the principles of population genetics to
prove a match mathematically
Your Own Personal Barcode?
We all like to think that we are unique, not like anyone else in the world. Unless
you are an identical twin, at the nuclear level, you are! Humans have 23 pairs of
chromosomes containing the DNA blueprint that encodes all the materials
needed to make up your body as well as the instructions for how to run it. One
member of each chromosomal pair comes from your mother, and the other is
contributed by your father.
Every cell in your body contains a copy of this DNA (see How Cells Work for
details). While the majority of DNA doesn't differ from human to human, some 3
million base pairs of DNA (about 0.10 percent of your entire genome) vary from
person to person. The key to DNA evidence lies in comparing the DNA left at the
scene of a crime with a suspect's DNA in these chromosomal regions that do
differ.
There are two kinds of polymorphic regions (areas where there is a lot of
diversity) in the genome:
• Sequence polymorphisms
• Length polymorphisms
Sequence Polymorphisms
Sequence polymorphisms are usually simple substitutions of one or two bases
in the genes themselves. Genes are the pieces of the chromosome that actually
serve as templates for the production of proteins. Amazingly, despite our
complexity, genes make up only 5 percent of the human genome. Individual
variations within genes aren't very useful for DNA fingerprinting in criminal cases.
Non-coding DNA
The other 95 percent of your genetic makeup doesn't code for any protein.
Because of this, these non-coding sequences used to be called "junk DNA,"
but it turns out that these regions do actually have important functions such as:
• Regulation of gene expression during development.
• Aiding or impeding cellular machinery from reading nearby genes and
making protein.
• Serving as the bricks and mortar of chromosomal structure.
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Length Polymorphisms
Non-coding DNA is full of length polymorphisms. Length polymorphisms are
simply variations in the physical length of the DNA molecule.
DNA evidence uses a special kind of length polymorphism found in non-coding
regions. These special variations come from stretches of short, identical repeat
sequences of DNA. A particular sequence can be repeated anywhere from one
to 30 times in a row, and so these regions are called variable number tandem
repeats (VNTRs).
The size of a DNA fragment will be longer or shorter, depending on how many
copies of a VNTR there are. In the case of DNA evidence, the great thing is that
the number of tandem repeats at specific places (called loci) on your
chromosomes varies between individuals. For any given VNTR loci in your DNA,
you will have a certain number of repeats.
You inherit one copy of each chromosome from your mother and father. This
means that you have two copies of each VNTR locus, just like you have two
copies of real genes. If you have the same number of sequence repeats at a
particular VNTR site, you are called homozygous at that site; if you have a
different number of repeats, you are said to be heterozygous.
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Creating a DNA Profile: The Basics
The basic procedure used to isolate an individual's DNA fingerprint is called
Restriction Fragment Length Polymorphism (RFLP) analysis. This is a
complicated way of saying that investigators determine the number of VNTR
repeats at a number of distinctive loci to come up with an individual's DNA profile.
Here is the key to DNA evidence: If you are looking at a particular person's
DNA, and a particular VNTR area in that person's DNA, there is going to be a
certain number of repeats in that area. What you do to make a DNA fingerprint is
to count the number of repeats for a specific person for a specific VNTR area.
For each person, there are two numbers of repeats in each VNTR region (one
from mom and one from dad), so you are getting both counts. If you do this for a
number of different VNTR regions, you can build a profile for a person that is
statistically unique. The resulting DNA fingerprint can then be compared with the
one left by the "perp" at a crime scene to see if there might be a match.
Here's how it works in general:
1. Isolate the DNA.
2. Cut the DNA up into shorter fragments containing known VNTR areas.
3. Sort the DNA fragments by size.
4. Compare the DNA fragments in different samples.
The way we sort by size is gel electrophoresis, and then we look at the results
using a Southern Blot
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Creating a DNA Profile: Step by Step
Now let's look at the exact steps used...
1. DNA is isolated from a sample such as blood, saliva, semen, tissue, or
hair. DNA has to be cleaned up, because, unlike in a pristine laboratory,
samples at a crime scene are often contaminated by dirt and other debris.
Sometimes, DNA must be isolated from samples dried to patches of cloth
or carpet, and getting the sample safely out of these fabrics adds
additional steps to the isolation and purification processes.
2. The huge genome is cut up with restriction enzymes to produce short,
manageable DNA fragments. These bacterial enzymes recognize specific
four to six base sequences and reliably cleave DNA at a specific base pair
within this span. Cleaving human DNA with one of these enzymes breaks
the chromosomes down into millions of differently sized DNA fragments
ranging from 100 to more than 10,000 base pairs long. You have to
carefully select an enzyme that doesn't cut within any of the VNTR loci
that are being studied; for RFLP analysis, the enzyme(s) chosen will
ideally cut close to the end on the outside of a VNTR region.
3. These DNA fragments are then sorted by size using gel electrophoresis.
In this process, DNA is loaded into a slab of Jell-O-like agarose and
placed in an electric field. The DNA is separated by size because:
DNA, being negatively charged, is pulled through the gel toward the
positively charged electrode.
Larger fragments move more slowly than smaller ones through the
porous agarose.
Once you have separated the DNA, you can determine the relative size of
each fragment based on how far it has moved through the agarose.
4. DNA fragments that have been separated on an agarose gel will begin to
disintegrate after a day or two. To permanently save the DNA fragments in
this segregated state, you need to transfer and permanently affix DNA to a
nylon membrane. First, the DNA is denatured from its native double helix
into a single-stranded state (this frees up nucleotides to base-pair with
DNA probes for step 5 of the process). The positively charged nylon
membrane is then placed on top of the agarose gel and used to sop up
the negatively charged DNA fragment, like you might blot ink off a
newspaper with Silly Putty.
5. Unlike Silly Putty, however, you can't actually see any of the DNA on your
membrane. In order to figure out which fragments contain a particular
VNTR locus, you have to flag them with some kind of tag that you can
visualize. How do you do this? You simply make use of the basic structure
and chemistry of DNA. DNA normally occurs as a double-stranded
molecule, as two strings of nucleotides twisted around each other. The
structure is held together by weak bonding between nucleotides on
opposing strands. Only certain pairs of nucleotides can interact (adenine
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with thymine and guanine with cytosine), so these nucleotides are said to
be complementary.
To locate a specific VNTR sequence on a single stranded DNA fragment,
you can find it by simply:
Making a DNA probe out of a DNA sequence complementary to
that of a VNTR locus
Labeling the probe with a radioactive compound
Letting the probe bind to like DNA sequences on the membrane
Using the radioactive tag to find where the probe has attached
6. Once the radioactive probe is stuck to its target on the membrane, you
can take a picture of it using special X-ray film. You don't need a camera
or other machinery to accomplish this feat --all you have to do is place the
membrane against a special sheet of film for a short period of time! How
does this work? In a regular camera, the film has a special coating that
undergoes chemical changes when it absorbs the energy of a photon of
light (to learn more, see How Photographic Film Works). When you take a
picture with a camera, the light you let in by opening the shutter forms an
image on the film. X-ray film, on the other hand, picks up radiation emitted
from the natural decay of the isotope used in your probe. What you see on
the film is a darkened band that indicates the places on the membrane
where the probe has bound to DNA containing the VNTR sequence.
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Courtesy Genelex
The results of RFLP analysis of one VNTR locus in
a sexual assault case
In the image above, DNA from suspects 1 and 2 are compared to DNA extracted
from semen evidence. You can see in this sample that suspect 1 and the sperm
DNA found at the scene match. Suspect 2 has a profile totally different from the
semen sample; his DNA fragments have run much farther down the gel, meaning
that they are shorter. You can also tell that he is a homozygote because there is
only one darker band indicating the presence of two copies of the same
fragment. The other samples tested come from heterozygotes, because they
have two bands of distinct sizes in each lane. DNA isolated from the victim as
well as a human DNA (K562) that serves as a standard size reference are
included as controls.
Crunching Numbers
The results from just one VNTR locus by itself don't pinpoint a suspect anymore
than having one digit of someone's Social Security number (SSN) would let you
figure out who they were. For example, a certain percentage of people are likely
to have the number 2 as the first digit in their SSN. Similarly, for any given VNTR
locus, a fragment length corresponding to a certain number of sequence repeats
occurs in a certain number of individuals. What gives DNA fingerprinting its
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power is the combined analysis of a number of VNTR loci located on different
chromosomes.
The final DNA profile is compiled from the results of four or five probes that are
applied to a membrane sequentially. Each probe targets a different VNTR locus.
Using four probes (as in the figure below) actually gives you eight pieces of
information about an individual, since each of us has two separate copies of each
VNTR region. To add to the complexity, it turns out that each VNTR locus
usually has approximately 30 different length variants (alleles). Each of these
alleles occurs at a certain frequency in a population. To get the probability that a
given 8 band profile will occur, you multiply the eight different allele frequencies
together.
Courtesy Genelex
While the number of repeats at a single VNTR locus
can't distinguish an individual from the rest of the
population, the combined results from a number of
loci produce a pattern unique to that person.
Using four loci, the probabilitythat you'd find a given allele combination in the
general population is somewhere around 1 in 5,000,000. In the United States,
the FBI incorporates 13 sites on average into its profiles. With 26 different bands
studied, you'd be incredibly hard pressed to find two unrelated individual with the
same DNA profile; the odds of a match in this case are well more than one in a
hundred billion. The bottom line is that, unless you have a twin, you're statistically
two thousand times more likely to win the Publisher's Clearinghouse
sweepstakes (1 in 50,000,000) than to have a DNA profile that matches anyone
else.
Advances in DNA Evidence
In 1985, DNA entered the courtroom for the first time as evidence in a trial, but it
wasn't until 1988 that DNA evidence actually sent someone to jail. This is a
complex area of forensic science that relies heavily on statistical predictions; in
early cases where jurors were hit with reams of evidence heavily laden with
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mathematical formulas, it was easy for defense attorneys to create doubt in
jurors' minds. Since then, a number of advances have allowed criminal
investigators to perfect the techniques involved and face down legal challenges
to DNA fingerprinting. Improvements include:
• Amount of DNA needed - RFLP analysis requires large amounts of
relatively high-quality DNA. Getting sufficient DNA for analysis has
become much easier since it became possible to reliably amplify small
samples using the polymerase chain reaction (PCR). With PCR, tiny
amounts of a specific DNA sequence can be copied exponentially within
hours.
• Source of DNA - Science has devised ingenious ways of extracting DNA
from sources that used to be too difficult or too contaminated to use.
• Expanded DNA databases - Several countries, including the U.S. and
Britain, have built elaborate databases with hundreds of thousands of
unique individual DNA profiles. This adds a lot of weight to arguments
formerly based on mathematical theory alone, but it does raise questions
of civil liberty as authorities ponder whether everyone in a community
should be forced to submit a sample for the sake of completeness.
• Training - Crime labs have come up with formal protocols for handling
and processing evidence, reducing the likelihood of contamination of
samples. On the courtroom side, prosecutors have become more savvy at
presenting genetic evidence, and many states have come up with specific
rules governing its admissibility in court cases.
• Science education - In recent years, a number of debates have erupted
around the world over issues like using DNA evidence, cloning animals or
selling genetically modified crops. It has dawned on many that to be
active, informed participants in such ethical debates, the general public
must understand the basic tenets of genetics, statistics, and the like.
Students in some schools today aren't just learning about dominant and
recessive genes in a lecture; they are performing PCR and RFLP analysis
on samples to look for that recessive gene!
Using DNA Evidence
Given the high profile DNA evidence had during the O.J. Simpson trial, most
people know DNA profiles are used by criminal investigators to:
• Prove guilt - Matching DNA profiles can link a suspect to a crime or crime
scene. The British police have an online database of more than 360,000
profiles that they compare to crime scene samples; more than 500 positive
matches come up a week.
• Exonerate an innocent person - At least 10 innocent people have been
freed from death row in the United States after DNA evidence from their
cases was studied. So far, DNA evidence has been almost as useful in
excluding suspects as in fingering and convicting them; about 30 percent
18

of DNA profile comparisons done by the FBI result in excluding someone
as a suspect.
DNA evidence is also useful beyond the criminal courtroom in:
• Paternity testing and other cases where authorities need to prove
whether or not individuals are related - One of the more infamous paternity
cases of late revolved around a 1998 paper in the journal "Nature" that
studied whether or not Thomas Jefferson, the third president of the United
States, actually fathered children with one of his slaves (in case you're
wondering, according the researchers, the answer is a resounding yes).
Photo courtesy Genelex, Inc
DNA evidence can pinpoint whether or not
someone is a parent.
• Identification of John or Jane Does - Police investigators often face the
unpleasant task of trying to identify a body or skeletal remains. DNA is a
fairly resilient molecule, and samples can be easily extracted from hair or
bone tissue; once a DNA profile has been created, it can be compared to
samples from families of missing persons to see if a match can be made.
The military even uses DNA profiles in place of the old-school dog tag.
Each new recruit must provide blood and saliva samples, and the stored
samples can subsequently be used as a positive ID for soldiers killed in
the line of duty. Even without a DNA match to conclusively identify a body,
a profile is useful because it can provide important clues about the victim,
such as his or her sex and race.
19

• Studying the evolution of human populations - Scientists are trying to
use samples extracted from skeletons and from living people around the
world to show how early human populations might have migrated across
the globe and diversified into so many different races.
• Studying inherited disorders - Scientist also study the DNA fingerprints
of families with members who have inherited diseases like Alzheimer's
Disease to try and ferret out chromosomal differences between those
without the disease and who are have it, in the hopes that these changes
might be linked to getting the disease.
Source: http://science.howstuffworks.com/dna-evidence.htm
20

II. Tools and Techniques for DNA Forensic Science
A. Detection of Body Fluids
Ultraviolet Light Illumination
Short wave 254nm/Long wave 365 nm
The use of ultraviolet (UV) light can be of great assistance in many forms of
forensic investigation. Since body fluids like semen, saliva, perspiration and
vaginal fluids are naturally fluorescent, the use of a UV light source offers a
unique method for locating these stains. The dried bodily fluids will naturally
fluoresce when illuminated with UV light source. We can then isolate the exact
location of the stain(s) to test, instead of testing the entire, large pieces of
evidence such as a mattress, a carpet, a sheet, an article of clothing, etc.
Microscopy
Individual sperm heads can be accurately identified based on their morphological
characteristics, using a phase contrast microscope and 20–100x magnification.
An ideal, mature spermatozoon has an oval shaped head with a regular contour
(4.0-5.0 um long and 2.5-3.5 um wide) with a pale anterior part (acrosome; 40-
70% of the head area) and a darker posterior region. The length-to-width ratio of
the head should be 1.50 to 1.75. The sperm tail should be attached in a
symmetrically situated fossa in the base of the head. The base of the head
should be broad and not arrow-like. Only one tail should be attached (about 45
um long), not coiled, nicked or bent over itself. Immediately behind the head the
first part of the tail, the mid piece, should be somewhat thicker (maximum width =
1 µm) and about 7-8 um long.
Source: http://www.dnatesting.biz/Semen_Sperm_ID/semen_sperm_id.html
21

Acid Phosphatase Test
One of the unique properties associated with semen is the presence of an
enzyme called prostatic acid phosphatase (PAP). PAP is not a single enzyme but
an array of related isoenzymes from a variety of sources. The PAP assay is a
well-documented presumptive assay for the presence of semen (1-4). Acid
phosphatase activity is 50-1000 times greater in human semen than in any other
bodily fluid. Unfortunately, the use of acid phosphatase as a marker for semen is
compromised because the vagina is also a source of vaginal acid phosphatase.
Since seminal and vaginal acid phosphatase can not discriminate, the only
approach to differentiating semen in vaginal secretion is by quantitative analysis.
Finding a significantly elevated acid phosphatase level is consistent with the
presence of semen. For example, if semen is present the acid phosphatase
assay is very robust and solution will immediately turn a deep purple color. If the
solution does not immediately turn purple or takes several minute to hour to turn
color then you are more than likely detecting endogenous vaginal acid
phosphatase and not semen.
Principle of enzyme-linked detection:
Source: http://www.dnatesting.biz/Semen_Sperm_ID/semen_sperm_id.html
22

p30 Test
Unlike the presumptive acid phosphatase test, the detection of the p30 antigen
requires the presence of the protein without the need of the target to perform an
enzymatic function. This aids in the identification of semen in aged evidence
samples in which the acid phosphatase enzyme is functionally inactive. Prostate
specific antigen (PSA, also known as p30), is a glycoprotein produced by the
prostatic gland and secreted into seminal plasma (fluid). The p30 is secreted in
seminal fluid at concentrations of 200,000 to 5.5 million ng per mL. The
sensitivity of the p30 test is 4 ng/mL and therefore seminal fluid diluted up to 1 in
a million can also be detected. This means we can detect semen in samples that
have been rinsed or washed (without detergent). In addition, p30 protein is
produced from a larger protein that is degraded to release the p30 protein. Since
the p30 is a product of protein degradation it is readily detected in very old
samples and sample that have been stored in a plastic bags for a long periond.
The detection of the p30 antigen in forensic samples is often helpful because it
confirms the presence of semen even in samples that involve vasectomized or
azospermic individuals. The reported frequency of azoospermia of 1-9% in
seminal stains or swabs examined in sexual assault cases (1) can be expected
to rise, since the frequency of contraceptive vasectomy has been estimated to be
750 000 to 1,000 000 per year in the United States (2).
Source: http://www.dnatesting.biz/Semen_Sperm_ID/semen_sperm_id.html
23

THE KASTLE-MEYER TEST
I. HISTORY
The Kastle-Meyer test got a huge start from Louis-Jacques Thenard and
Christian Freidrich Schonbein. Thenard discovered hydrogen peroxide in 1818,
and Schonbein developed one of the first presumptive tests in 1863. (Source:
http://www.forensicdna.com/Timeline.htm ) This test was based on the
observation that the peroxidase-like activity in hemoglobin causes oxidation of
hydrogen peroxide. The result of the reaction between hydrogen peroxide and
hemoglobin is the appearance of “foaming” as the oxygen bubbles rise. (Perhaps
you have seen this reaction yourself when washing out a cut with peroxide.)
Shonbein reasoned that if an unknown stain foamed when hydrogen peroxide
was applied to it, then that stain probably contained hemoglobin, and therefore
was likely to be blood.
Schonbein
Louis-Jacques Thenard Christian Freidrich
In the early 1900’s, Dr. Kastle developed a presumptive test for hemoglobin
which used phenolphthalein (feen-awl-THAY-leen) as a color indicator. A few
years later, Dr. Meyer refined and improved upon this test, and this is why it is
sometimes called the Kastle-Meyer test.
II. STRENGTHS AND LIMITATIONS
When discussing various presumptive tests, it is useful to know two terms in
particular: sensitivity and specificity. Sensitivity refers to the dilution factor of
a substance (in this case, blood) that can still be detected by the test. The
sensitivity of the KM test is approximately 1:10,000. This means that if one drop
of blood were added to ten thousand drops of water, the KM test would still give
a reaction to that drop of blood!
24

Specificity refers to how many other substances the test will react to other than
blood. So far, a true positive reaction has been observed to occur only in the
presence of hemoglobin. False-positives may occur in the presence of chemical
oxidants, and it is possible for vegetable-peroxidases to react with the test too,
but these reactions would occur after phenolphthalein was applied and before the
addition of hydrogen peroxide. A true positive result develops only after the
hydrogen peroxide is applied. For this reason, it can be said that the KM test is
highly specific for blood.
III. THE KM TEST: UP CLOSE AND PERSONAL
Let’s examine the items contained in a typical test kit to see what they’re for.
1.) Positive Control: This is a small swatch of cloth that is stained with
dried, non-human blood. (Animal blood is used to stain the control in order
to prevent the possible transmission of diseases.) It is included for the
purpose of serving as a known sample of blood so that the reagents can
be checked before beginning a series of tests at a scene. It would be very
unprofessional if you were to test stains at a crime scene without checking
your reagents first. What if your tests indicated that there was no blood
present only because your phenolphthalein had become oxidized in the
bottle (maybe because the cap wasn’t on very tight, for instance)? Always
check your reagents with the positive control before doing any actual
testing!
2.) Transfer Materials: These may include cotton swabs, filter papers, or, if
you need to get into really small spaces, cotton string or thread. You’ll be
transferring some of the suspect stain onto these media for testing, NOT
applying the test directly onto the evidential stain!! (This is why KM is
considered an indirect test.)
3.) The Reagents: There are three reagents included with the kit. These
are:
a. Alcohol: Methyl or Ethyl alcohol is used to increase the
sensitivity of the test. It does this by “cleaning up” the area in and
around the bloodstain to better expose the hemoglobin.
b. Phenolphthalein: This is a solution which acts as a color
indicator. When prepared, the solution is boiled for several hours to
help remove most of the oxygen trapped in it. It should appear as a
25

colorless liquid. When this solution is oxidized (exposed to oxygen),
it will turn pink.
c. Hydrogen Peroxide: This is the 3% form typically found in
drugstores. Hydrogen peroxide is essentially water with an extra
oxygen atom attached to it. You can think of it as a chemical
oxidant which is contained in your kit.
4.) Deionized (D.I.) Water: While not an actual reagent, D.I. water is
sometimes included with a test kit in order to facilitate the transfer of stain
material onto your swab, filter paper, etc.
IV. APPLICATION OF THE KM TEST
Let’s assume that you’ve completed your visual examination of the scene and /
or evidentiary items, and 1) found some stains of interest or 2), had reason to
suspect that blood might be present. How is this test applied?
Before beginning, keep in mind the importance of avoiding contamination! You
should have the necessary personal protective equipment in place (gloves, etc.),
and be cautious about how you use the materials in the kit. Do not touch the
dropper-bottle tips with the swabs, try not to switch the caps of one reagent with
another, always use a fresh swab for each test, etc. Do not apply reagents
directly over the evidence please!
You’ll begin by performing a test with the positive control. A drop or two of deionized
(D.I.) water is applied to the swab to moisten it, and then the swab is
rubbed lightly against the control. Once this is done, begin applying the reagents
in the following manner:
1.) Alcohol: Apply a drop or two onto your swab.
2.) KM: Apply a drop or two onto the swab.
At this point you will need to pause for a few seconds and look for any
sign that the swab is beginning to develop a pink color. This is not
26

expected to occur when using your control, but if it is, you are seeing a
false-positive reaction and should not continue further. Something is
interfering with the test. If no color is seen developing, proceed with the
last step which is:
3.) Hydrogen peroxide: Apply a drop or two onto the swab. One should
see a pink color develop almost immediately.
The control need only be performed once before beginning a series of tests.
Thereafter, repeat the same procedure throughout testing of suspect stains.
V. INTERPRETATION OF RESULTS
Presumptive tests can give very reliable indications of blood’s presence, but
cannot be interpreted to mean that blood is absolutely identified. Only
confirmatory tests can do this. The table below describes various reactions
possible with this test and the proper interpretations that can be made from them:
PINK COLOR
DEVELOPS..
RESULT INTERPRETATION
After KM is applied False positive The reaction did not occur
as the result of blood’s
presence.
After hydrogen peroxide
is applied
Positive Blood is indicated
Never
Negative
False negative
27
Blood is not indicated
(may not be present)
Blood may be present, but
is too dilute to react

VI. AN IMPORTANT NOTE
One should not “read” the result of the KM test after 30 seconds or so. This
is because the KM reagent may begin to turn pink after this time as the result of
oxygen in the ambient environment, and not because blood is present.
VII. HOW DOES THIS TEST WORK?
The diagram below helps illustrate what happens in the KM test. Follow along as
I describe each part.
1.) Hydrogen peroxide: Look at the chemical formula and compare it with the
chemical formulas for water and oxygen. Oxygen (elemental oxygen) typically
comes as a pair of molecules (as it has in the peroxide), but something in our test
breaks it up. What might that be?
2.) Heme: This is where we find peroxidase-like activity. This is what breaks up
peroxides. Peroxides are toxic to animal tissue, and are decomposed by the
heme in our blood. In this case, hydrogen peroxide is decomposed into two
parts: water, and a free oxygen radical.
3.) Follow the O (the free radical). It's going to combine with the KM, our
phenolphthalein color-indicator. Why?
4.) When the KM solution is initially prepared, it is boiled in a flask for several
hours to help remove oxygen. (A little bit of zinc metal dust added to the solution
28

helps to bind oxygen as well.) When the solution turns colorless, that means the
KM has had most of its oxygen removed, and is now "oxygen hungry". So... now
we have the makings of a kind of love story: the KM is badly in want of oxygen,
and there's a lonely oxygen radical floating around, un-spoken for... What do you
suppose happens next?
5.) Pink color: This is the blushing that occurs when the oxygen radical "hooks
up" with the KM reagent. The radical oxidizes the KM, and this oxidation causes
the KM to turn pink.
Source: http://www.geocities.com/a4n6degener8/kastle.htm
29

Luminol
What Does Luminol Do?
Much of crime scene investigation, also called criminalistics, is based on the
notion that nothing vanishes without a trace. This is particularly true of violent
crime victims. A murderer can dispose of the victim's body and mop up the pools
of blood, but without some heavy-duty cleaning chemicals, some evidence will
remain. Tiny particles of blood will cling to most surfaces for years and years,
without anyone ever knowing they're there.
The basic idea of luminol is to reveal these traces with a light-producing chemical
reaction between several chemicals and hemoglobin, an oxygen-carrying protein
in the blood. The molecules break down and the atoms rearrange to form
different molecules (see Microsoft Encarta: Chemical Reaction for more
information on chemical reactions). In this particular reaction, the reactants (the
original molecules) have more energy than the products (the resulting
molecules). The molecules get rid of the extra energy in the form of visible light
photons. This process, generally known as chemiluminescence, is the same
phenomenon that makes fireflies and light sticks glow.
A simulation of luminol at work: Before spraying
luminol, there's no sign of blood. After spraying
luminol, the latent blood traces emit a blue glow.
Investigators will spray a suspicious area, turn out all the lights and block the
windows, and look for a bluish-green light. If there are any blood traces in the
area, they will glow.
The Chemical Reaction
The "central" chemical in this reaction is luminol (C8H7O3N3), a powdery
compound made up of nitrogen, hydrogen, oxygen and carbon. Criminalists mix
the luminol powder with a liquid containing hydrogen peroxide (H2O2), a
hydroxide (OH-) and other chemicals, and pour the liquid into a spray bottle. The
hydrogen peroxide and the luminol are actually the principle players in the
30

chemical reaction, but in order to produce a strong glow, they need a catalyst to
accelerate the process. The mixture is actually detecting the presence of such a
catalyst, in this case the iron in hemoglobin (see Microsoft Encarta: Catalysis for
more information on catalysts).
To perform a luminol test, the criminalists simply spray the mixture wherever they
think blood might be. If hemoglobin and the luminol mixture come in contact, the
iron in the hemoglobin accelerates a reaction between the hydrogen peroxide
and the luminol. In this oxidation reaction, the luminol loses nitrogen and
hydrogen atoms and gains oxygen atoms, resulting in a compound called 3aminophthalate.
The reaction leaves the 3-aminophthalate in an energized state -
- the electrons in the oxygen atoms are boosted to higher orbitals. The electrons
quickly fall back to a lower energy level, emitting the extra energy as a light
photon (see How Fluorescent Lamps Work for more information on light
production). With iron accelerating the process, the light is bright enough to see
in a dark room. Investigators may use other chemiluminescent chemicals, such
as fluorescein, instead of luminol. These chemicals work the same basic way, but
the procedure is a little bit different.
How Investigators Use Luminol
If luminol reveals apparent blood traces, investigators will photograph or
videotape the crime scene to record the pattern. Typically, luminol only shows
investigators that there might be blood in an area, since other substances,
including household bleach, can also cause the luminol to glow. Experienced
investigators can make a reliable identification based on how quickly the reaction
occurs, but they still need to run other tests to verify that it is really human blood.
Luminol in itself won't usually solve a murder case. It's only one step in the
investigative process. But it can reveal essential information that gets a stalled
investigation going again. For example, hidden blood spatter patterns can help
investigators locate the point of attack and even what sort of weapon was used
(a bullet makes blood splatter very differently than a knife does). Luminol may
also reveal faint bloody shoe prints, which gives investigators valuable
information about the assailant and what he or she did after the attack.
In some cases, luminol leads investigators to more evidence. For example, if
luminol detects trace amounts of blood on a carpet, investigators may pull up the
carpet and discover a lot of visible blood on the floorboards below.
One problem with luminol is that the chemical reaction can destroy other
evidence in the crime scene. For this reason, investigators only use luminol after
exploring a lot of other options. It is definitely a valuable tool for police work, but
it's not quite as prevalent in crime investigation as presented on some TV shows.
The police don't walk into a crime scene and start spraying luminol on every
visible surface. Source: www.howstuffworks.com
31

Saliva Evidence
Amylase activity: The most common tests for the identification of saliva are
dependent upon the detection of amylase (1-3). Confirmation of amylase means
that results are consistent with saliva and then consistent with oral sex. A high
level of amylase activity is usually indicative of the presence of saliva. With the
exception of feces, no other body fluid approaches the level of amylase activity in
dried stains. Amylase is a very stable enzyme and can be detected even on
dried articles.
Salivary amylase: As with most enzymes, the function of salivary amylase can
be deduced from the name. "Salivary" refers to the fact that it is found in saliva.
The suffix "ase" is usually added to the end of enzymes as an indication that the
name refers to an enzyme. Amylose is a kind of starch in which glucose
monomers are joined by α(1—4) linkages. α-Amylase (α-1,4-glucan 4glucanohydrolase,
EC 3.2.1.1) is an enzyme that degrades starch to
oligosaccharides and in turn to maltose and glucose by hydrolyzing α-1,4-glucan
bonds. In digestion, the role of α-amylase is primarily the first reaction of this
process, generating oligosaccharides that are then hydrolyzed by other enzymes.
Salivary amylase is very specific in the way it breaks down starch to glucose. It
inserts a water molecule at the α(1—4) linkage closest to the end of the amylose
molecule thus releasing a glucose molecule. We can measure this break down
to determine if salivary amylase is present (see figure below). Positive amylase
tests confirm amylase activity, which is consistent with saliva.
The amylase assay is very sensitive and can detect amylase activity down to a
final concentration of 1 x 10 -4 U/mL (0.001 units). Amylase concentration in
human saliva is 42–59 U/mL (4). One unit (U) is defined as the amount of
enzyme required to liberate 1 mg of maltose from starch in 3 minutes at 20° C, at
pH 6.9.
The mechanism of α-amylase. The enzyme protonates (hydrolizes) the acetal
linkage between the n-sugar residue (sugar on the left) and the rest of the
saccharide chain to yield a free glucose residue to convert to energy.
References
1. Brauner, P., "The Evidence Notwithstanding--A case Report on a Rape,"
Journal of Forensic Sciences, JFSCA, Vol. 37, No.1, Jan. 1992, pp.345-348
32

2. Brauner, P.& Gallili, N., "A Condom--the Critical Link in a Rape," Journal of
Forensic Sciences, JFSCA, Vol. 38, No.5, September 1993, pp.1233-1236.
3. "Forensic Science Symposium On The Analysis of Sexual Assault Evidence",
Proceedings, Forensic Science Research and Training Center, Laboratory
Division, FBI Academy, Quantico, Virginia, 1983, July 6-8
4. McCloskey, K.L. & Muscillo, G.C. & Noordewier, B., "Prostatic Acid
Phosphatase Activity in the Postcoital Vagina," Journal of Forensic Sciences,
1975, vol.20, p.630-636
Source: http://www.dnatesting.biz/Semen_Sperm_ID/semen_sperm_id.html
33

B. Isolation of DNA
Differential Lysis
Once a suspected sample has been identified to contain sperm it is often
contaminated with other cell types. The most common contaminating cells are
the epithelial cells lining the vaginal wall, but can include epithelial cells from the
mouth (buccal cells) and skin, as well as those found in urine.
DNA from contaminating epithelial cells can be removed using a procedure
called a differential extraction (5), which takes advantage of unique properties
associated with each cell type. In this procedure, the cells are removed from the
suspected material by soaking them in a gentle solution. Epithelial cell DNA is
isolated under mild conditions that break open the epithelial cells but leave the
sperm cells intact DNA. The DNA in sperm can then be extracted using a more
harsh extraction procedure.
Y chromosome: Since the differential lysis procedure is unsuitable for saliva
evidence, the ratio of male to female cells becomes the deciding factor in cases
of sexual infidelity. For neat saliva stains or body surface swabs, it can be
possible to generate a clean or dominant male type using current STR
technology. For vaginal secretions it becomes more difficult to show the
presence of male DNA with in a sea of female DNA. Y-STR testing can help to
verify the presence of male DNA were infidelity involving oral sex is considered.
References:
5. Gill, P., Jeffreys, A.J., Werret, D.J., Nature 1985, 318, 577-579
Source: http://www.dnatesting.biz/Semen_Sperm_ID/semen_sperm_id.html
34

DNA Extraction Techniques
Organic-based Extraction
Phenol extraction is a common technique used to purify a DNA sample.
Generally, samples are extracted by addition of one-half volume of neutralized
(with TE buffer, pH 7.5) phenol to the sample, followed by vigorous mixing for a
few seconds to form an emulsion. Following centrifugation for a few minutes, the
aqueous (top) phase containing the nucleic acid is recovered and transferred to a
clean tube. Residual phenol then is removed by extraction with an equal volume
of water-saturated diethyl ether. Following centrifugation to separate the phases,
the ether (upper) phase is discarded and the DNA is concentrated by ethanol
precipitated.
Chelex
Chelex extraction is used to isolate the DNA away from the rest of the contents
of the cells. The protocol consists of mixing cells with 5% chelex and Proteinase
K and incubating the solution at 56 o C. The proteinase K is an enzyme that
digests proteins (cell membranes for example). The chemical chelex, in the form
of minute beads, binds to most of the contents of the cell except DNA. It does
this by chelating magnesium. A boiling step has been added to further breakup
the cells in order to free the DNA. The chelex beads (with the non-DNA cellular
components) are then removed by centrifugation leaving behind the DNA. This
DNA is single-stranded and ready for the polymerase chain reaction (PCR).
Silica
This extraction method consists of flowing a prepared DNA sample over silica
micro beads that attract and capture the DNA strands onto the beads. Upon
capture of the DNA strands, the DNA is washed and collected. In the presence of
high salt DNA binds to silica particles then the silica with adsorbed DNA washed
to remove salt and impurities from the original sample, and the clean DNA eluted
in water or buffer.
Silica extraction works with a wide size range of DNA and allows efficient
recovery (90%) of product. Using silica you can purify DNA as small as 100bp.
There is no upper size limit on recovery efficiency of DNA. However, precautions
should be taken during purification of longer DNA fragments (20kb) to avoid
shearing. Proteins and RNA do not bind to silica and are eliminated during
washes and for this reason it is also an ideal tool to purify and concentrate DNA
directly from various reaction mixtures. Because silica does not bind
oligonucleotides with high efficiency (

• purify DNA from any type of agarose;
• concentrate DNA (for changing buffer, desalting);
• remove proteins (after restriction enzyme treatment; dephosporylation with
BAP or CIAP);
• remove unincorporated nucleotides, primer, primer-dimers and enzymes
from a labeling reaction or PCR;
• remove an excess of linkers after ligation;
• remove residual phenol, chloroform, and ethidium bromide.
• purify plasmid DNA free of RNA from bacterial lysates.
Source: http://herpesvirus.tripod.com/research/protoDNA.htm
36

C. Quantification of DNA
Southern Blot
The Southern blot is used to detect the presence of a particular bit of DNA in a
sample. The DNA detected can be a single gene, or it can be part of a larger
piece of DNA such as a viral genome.
DNA is extracted from the cells and purified
Restriction Digest
DNA is restricted (cut) with enzymes.
DNA fragments
In this example, a large piece of DNA is chopped into
smaller pieces using a restriction enzyme.
Loading the Gel
The DNA is loaded into a well of the gel matrix.
• Lane 1 contains size standards (a mix of known DNA
fragments)
• Lane 2 contains the restricted DNA
• Lane 3 contains unrestricted (whole) DNA
37

Running the Gel
An electric current is passed through the gel and the DNA
moves away from the negative electrode. The distance
moved depends on the size of the DNA fragment. Standards
are used to quantitate the size. Unrestricted, large DNA runs
as a smear due to random shearing (breaking) of the DNA.
The DNA can be visualized by staining first with a
fluorescent dye and then lighting with UV.
Transfer of the DNA to a Membrane
The DNA is first denatured (made single stranded - usually
by raising the pH) and then transferred out of the gel and
onto a membrane. The transfer can be done electrically or
by capillary action with a high salt solution.
Development of the blot
A radioactively-labeled probe specific for the gene in
question is incubated with the blot. The blot is washed to
remove non-specifically bound probe and then a
development step allows visualization of the DNA that is
bound. See below for a detailed description of this process.
In this example the gene is found in the second largest
fragment of the restricted DNA. In the unrestricted DNA it
migrates more slowly because it is part of a larger molecule.
To use this method to quantify DNA it is necessary to compare your results to a
standard curve. You would need to use a smaple that contains a known quantity
of DNA.
38

Southern Up Close
In this simplified cartoon, two different DNAs
(single stranded following denaturation) are
bound to a membrane.
The membrane is incubated with a solution
containing a probe (another single stranded DNA
or RNA, or oligonucleotide) which is homologous
to one of the two DNAs on the membrane. The
probe has an indicator attached to it so that it
may be detected later on. The binding is very
specific and requires the base pairs of the probe
and the bound DNA to be perfectly
complementary, i.e. A to T and C to G or for
RNA; A to U and C to G.
A wash step removes any probe which is not
tightly bound to the DNA on the membrane. Only
DNA that matches exactly will remain bound.
In this example, DNA "A" is the specific DNA
homologous to the probe and therefor a band
will develop where this DNA is bound to the
membrane. No band will show where "B" is
bound
source: http://www.escience.ws/b572/L22/south.html
39

Real-Time Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) allows the logarithmic copying of part of
a DNA molecule using a DNA polymerase enzyme that is tolerant to
elevated temperatures. RT PCR uses mRNA as a template to make
DNA. The steps involved are discussed below:
1. mRNA is copied to cDNA (chromosomal DNA) by reverse transcriptase
using an oligo dT primer (random oligomers may also be used). In realtime
PCR, we usually use a reverse transcriptase that has an endo H
activity. This removes the mRNA allowing the second strand of DNA to be
formed. A PCR mix is then set up which includes a heat-stable
polymerase (such as Taq polymerase), specific primers for the gene of
interest, deoxynucleotides and a suitable buffer.
40

2. cDNA is denatured at more than 90 degrees (~94 degrees) so that the
two strands separate. The sample is cooled to 50 to 60 degrees and
specific primers are annealed that are complementary to a site on each
strand. The primers sites may be up to 400 bases apart but are often
about 100 bases apart, especially when real-time PCR is used.
3. The temperature is raised to 72 degrees and the heat-stable Taq DNA
polymerase extends the DNA from the primers. Now we have four cDNA
strands (from the original two). These are denatured again at
approximately 94 degrees.
41

This process is repeated many times.
After 30 to 40 rounds of synthesis of cDNA, the reaction products are
usually analyzed by agarose gel electrophoresis. The gel is stained with
ethidium bromide to visualize the cDNA.
An agarose gel stained with ethidium bromide
and illuminated with UV light which causes the intercalated stain to
fluoresce.
This type of agarose gel-based analysis of cDNA products of reverse
transcriptase-PCR does not allow accurate quantitation since ethidium
bromide is rather insensitive and when a band is detectable, the
logarithmic stage of amplification is over. This problem will be addressed
in more detail below.
Ethidium bromide is a dye that binds to double stranded DNA by
interpolation (intercalation) between the base pairs. Here it fluoresces
when irradiated in the UV part of the spectrum. However, the fluorescence
is not very bright. Other dyes such as SYBR green, which are much more
fluorescent than ethidium bromide, are used in real time PCR.
42

SYBR-green
SYBR green is another method to monitor DNA synthesis. SYBR green is
a dye that binds to double stranded DNA, but not to single-stranded DNA,
and is frequently used to monitor the synthesis of DNA during real-time
PCR reactions. When it is bound to double stranded DNA it fluoresces
very brightly (much more brightly than ethidium bromide does). We also
use SYBR green because the ratio of fluorescence in the presence of
double-stranded DNA to the fluorescence in the presence of singlestranded
DNA is much higher that the ratio for ethidium bromide. Other
methods can be used to detect the product during real-time PCR.
Sybr-green fluoresces brightly only when bound to double-stranded DNA
There are many real time machines available and the one shown below is
the ICycler ® from BioRad. The lid slides back to accommodate samples in
a 96-well plate format. This means that we can look at a lot of samples
simultaneously. The machine contains a sensitive camera that monitors
the fluorescence in each well of the 96-well plate at frequent intervals
during the PCR Reaction. As DNA is synthesized, more SYBR Green will
bind and the fluorescence will increase.
source: http://www.med.sc.edu:85/pcr/realtime-home.htm
43

D. Creating a DNA Profile
RFLP Method - Restriction Fragment Length Polymorphism
RFLP (often pronounced "rif lip", as if it were a word) is a method used by
molecular biologists to follow a particular sequence of DNA as it is passed on to
other cells. RFLPs can be used in many different settings to accomplish different
objectives. Some uses include in paternity cases or criminal cases to determine
the source of a DNA sample, to determine the disease status of an individual, or
to measure recombination rates which can lead to a genetic map with the
distance between RFLP loci measured in centiMorgans.
RFLP Production
Each organism inherits its DNA from its parents. Since DNA is replicated with
each generation, any given sequence can be passed on to the next generation.
An RFLP is a sequence of DNA that has a restriction site on each end with a
"target" sequence in between. A target sequence is any segment of DNA that
bind to a probe by forming complementary base pairs. A probe is a sequence of
single-stranded DNA that has been tagged with radioactivity or an enzyme so
that the probe can be detected. When a probe base pairs to its target, the
investigator can detect this binding and know where the target sequence is since
the probe is detectable. RFLP produces a series of bands when a Southern blot
is performed with a particular combination of restriction enzyme and probe
sequence.
For example, let's follow a particular RFLP that is defined by the restriction
enzyme EcoR I and the target sequence of 20 bases
GCATGCATGCATGCATGCAT. EcoR I binds to its recognition seuqence
GAATTC and cuts the double-stranded DNA as shown:
In the segement of DNA shown below, you can see the elements of an RFLP; a
target sequence flanked by a pair of restriction sites. When this segment of DNA
is cut by EcoR I, three restriction fragments are produced, but only one contains
the target sequence which can be bound by the complementary probe
sequence(purple).
44

Let's look at two people and the segments of DNA they carry that contain this
RFLP (for clarity, we will only see one of the two stands of DNA). Since Jack and
Jill are both diploid organisms, they have two copies of this RFLP. When we
examine one copy from Jack and one copy from Jill, we see that they are
identical:
Jack 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---
GAATTC-
Jill 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---
GAATTC-
When we examine their second copies of this RFLP, we see that they are not
identical. Jack 2 lacks an EcoR I restriction site that Jill has 1.2 kb upstream of
the target sequence (difference in italics).
Jack 2: -GAATTC--(1.8 kb)-CCCTTT--(1.2 kb)--GCATGCATGCATGCATGCAT--
(1.3 kb)-GAATTC-
Jill 2: -GAATTC--(1.8 kb)-GAATTC--(1.2 kb)--GCATGCATGCATGCATGCAT--
(1.3 kb)-GAATTC-
Therefore, when Jack and Jill have their DNA subject to RFLP analysis, they will
have one band in common and one band that does not match the other's in
molecular weight:
45

Paternity Case
Let's use RFLP technology to determine if Jack is the father of Jill's child named
Payle.
In this scenario, DNA was extracted from white blood cells from all three
individuals and subjected to RFLP analysis. The results are shown below:
In this case, it appears that Jack could be the father, since Payle inherited the
12.4 kb fragment from Jill and the 4.3 fragment from Jack. However, it is possible
that another man with similar RFLP pattern could be as well.To be certain,
several more RFLP loci would be tested. It would be highly unlikely that two men
(other than identical twins) would share multiple RFLP patterns and so paternity
could be confirmed.
In a different scenario, DNA was extracted from white blood cells from all three
individuals and subjected to RFLP analysis. The results are shown below:
This time, it can be determined that Jack is NOT the father of Payle since Payle
has a band of about 6 kb and Jack does not. Therefore, it is very probable that
Payle's father is not Jack, though it is possible that Payle carries a new mutation
46

at this locus and a different sized band was produced. What could you do as an
investigator to be more certain that Jack was not the father of Payle?
Disease Status
In this example, we want to know if a person carries any cystic fibrosis (CF)
alleles and if so, how many. Because CF is a recessive disease, anyonne with
CF must be homozygous for disease alleles. From pedigree information, we can
often determine who in this family is a carrier. However, if a couple comes to a
genetic counselor, often an RFLP analysis is performed on the couple's DNA.
RFLPs are known for CF and so it would be easy to determine if a person were
homozygous wild-type (wt), heterozygous "carrier", or homozygous disease
alleles and thus have CF.
For couples expecting a child, it would be simple to test both parents and make a
prediction about the eventual disease status of their fetus. For example, if both
parents were homozygous wt, then all of their children would also be
homozygous wt:
47

However, if both parents were heterozygous, they could have children with any of
the three genotypes, though heterozygous children would be twice as likely as
either of the homozygous genotypes.
With increasing genomic sequence information, increasing numbers of genetic
disease can be predicted from RFLP analyses.
Genetic Mapping
To calculate the genetic distance between to loci, you need to be able to observe
recombination. Traditionally, this was performed by observing phenotypes but
with RFLP analysis, it is possible to measure the genetic distance between two
RFLP loci whether they are a part of genes or not.
Let's look at a simple example in fruit flies. Two RFLP loci with two RFLP bands
possible at each locus:
48

These loci are located on the same chromosome for the female (left) and the
male (right). The upper locus can produce two different bands called 1 and 3.
The lower locus can produce bands called 2 or 4. The male is homozygous for
band 1 at the upper locus and 2 for the lower locus. The female is heterozygous
at both loci. Thier RFLP banding patterns can be seen on the Southern blot
below:
The male can only produce one type of gamete (1 and 2) but the female can
produce four different gametes. Two of the possible four are called parental
because they carry both RFLP bands from the same chromosome; 1 and 2 from
the left chromosome or 3 and 4 from the right chromosome. The other two
chromosomes are recombinant because recombination has occurred between
the two loci and thus the RFLP bands are mixed so that 1 is now linked to 4 and
3 is linked to 2.
49

When these two flies mate, the frequency of the four possible progeny can be
measured and from this information, the genetic distance between the two RFLP
loci (upper and lower) can be determined.
In this example, 70% of the progeny were produce from parental genotype eggs
and 30% were produced by recombinant genotype eggs. Therefore, these two
RFLP loci are 30 centiMorgans apart from each other.
Source: http://www.bio.davidson.edu/courses/genomics/method/RFLP.html
50

Short Tandem Repeat (STR) Analysis
Short tandem repeat (STR) technology is used to evaluate specific regions (loci)
within nuclear DNA. Variability in STR regions can be used to distinguish one
DNA profile from another. STR loci consist of short, repetitive sequence
elements 3 to 7 base pairs in length . These repeats are well distributed
throughout the human genome and are a rich source of highly polymorphic
markers, which may be detected using the polymerase chain reaction (5–8).
Alleles of STR loci are differentiated by the number of copies of the repeat
sequence contained within the amplified region and are distinguished from one
another using radioactive, silver stain or fluorescence detection following
electrophoretic separation.
One system, The PowerPlex® 16 System, allows the coamplification and threecolor
detection of sixteen loci (fifteen STR loci and Amelogenin). The loci are
grouped into three different group, each group labeled with a different compound
(fluorescein, JOE, and TMR) that will fluoresce either green, red, or blue. The
labeling occurs during PCR amplification of the DNA.
All sixteen loci are amplified simultaneously in a single tube and analyzed in a
single injection or gel lane. For more details, see below.
The Promega PowerPlex 16 system:
The fluorescein-labeled allelic ladder showing all possible repeat lengths in each
STR loci. The numbers on the ruler at the top (100-480) represent the number of
base pairs and the overall region where the loci is located. The boxes just blow
the ruler show the names of each loci. The peaks show all possible peaks for
each loci and the boxed in numbers below the peaks show the number of repeats
in each peak represents.
51

For example, in the first loci (which spans the region from 100-150 base pairs)
the victim could have 15 sequence repeats from her mother and 16 from her
father, while, the suspect could have 20 repeats from his mother and 18 from his
father. The PCR reaction with the fluorescein-labeled primers would have
attached tags onto each length of repeats. When the sample is run through
electrophoresis, the fluorescein tag would be illuminated, and a reading would be
taken. As we know, smaller samples run faster through electrophoresis, thus the
lane containing the victim’s DNA (which contains some 15-repeat segments and
some 16-repeat segments) would fluoresce and give a reading before the lane
containing the suspect’s DNA (containing 18- and 20-repeat segments).
The victim’s results would show two peaks for the first loci, one at 15 and one at
16. The suspect’s results would also show two peaks for the first loci, but they
would be at 18 and 20. Each of the five loci shown above would contain two
peaks for both the victim’s results and the suspect’s results.
………………………………………………………………….
100 120 140 160
15
16
…………………………………………………………………..
100 120 140 160
18
52
20
Victim’s results
Suspect’s
results

The other two PowerPlex16 groups of loci:
The JOE-labeled allelic ladder components
The TMR-labeled allelic ladder components
53

A sample of actual results from one person:
The top row is labeled with fluorescein, the middle with JOE, and the bottom with
TMR. The names at the top of each pair of peaks is the name of that particular
loci. Notice that each labeling group has five loci, and that each loci has two
peaks (one repeat segment from the mother and one from the father). To find
out how many repeats each peak represents, the results must be compared
against the allelic ladders shown above. The ruler at the very top represents the
number of base pairs, as do the peaks at the very bottom.
Source: www.promega.com
54

The Federal Bureau of Investigation (FBI) uses a standard set of 13 specific STR
regions for CODIS. CODIS is a software program that operates local, state, and
national databases of DNA profiles from convicted offenders, unsolved crime
scene evidence, and missing persons. The odds that two individuals will have the
same 13-loci DNA profile is about one in one billion.
DNA Forensics Databases
National DNA Databank: CODIS
The COmbined DNA Index System, CODIS, blends computer and DNA
technologies into a tool for fighting violent crime. The current version of CODIS
uses two indexes to generate investigative leads in crimes where biological
evidence is recovered from the crime scene. The Convicted Offender index
contains DNA profiles of individuals convicted of felony sex offenses (and other
violent crimes). The Forensic index contains DNA profiles developed from crime
scene evidence. All DNA profiles stored in CODIS are generated using STR
(short tandem repeat) analysis.
CODIS utilizes computer software to automatically search its two indexes for
matching DNA profiles. Law enforcement agencies at federal, state, and local
levels take DNA from biological evidence (e.g., blood and saliva) gathered in
crimes that have no suspect and compare it to the DNA in the profiles stored in
the CODIS systems. If a match is made between a sample and a stored profile,
CODIS can identify the perpetrator.
This technology is authorized by the DNA Identification Act of 1994. All 50 states
have laws requiring that DNA profiles of certain offenders be sent to CODIS. As
of January 2003, the database contained more than a million DNA profiles in its
Convicted Offender Index and about 48,000 DNA profiles collected from crime
scenes but which have not been connected to a particular offender.
As more offender DNA samples are collected and law enforcement becomes
better trained and equipped to collect DNA samples at crime scenes, the backlog
of samples awaiting testing throughout the criminal justice system has increased
dramatically. In March 2003 President Bush proposed $1 billion in funding over 5
years to reduce the DNA testing backlog, build crime lab capacity, stimulate
research and development, support training, protect the innocent, and identify
missing persons. For more information, see the U.S. Department of Justice's
Advancing Justice Through DNA Technology:
http://www.usdoj.gov/ag/dnapolicybooktoc.htm
55

E. Other Issues
Mitochondrial DNA (mtDNA)
The cell contains two kinds of DNA, a nuclear DNA genome (nucDNA) and
mitochondrial DNA (mtDNA). Mitochondrial DNA is the genetic information
present in cellular organelles known as mitochondria. These organelles are
present in the cytoplasm of the cell and they provide most of the energy a cell
needs. One mitochondrion has several copies of mtDNA and there are over fifty
mitochondria per cell. Thus a cell could have hundreds of mtDNA.
Since mtDNA is inherited from one generation to the next exclusively from the
mother, every individual within a given maternal lineage should have the same
mtDNA. The figure below demonstrates this inheritance pattern for mtDNA.
Similar colours indicate an identical mtDNA pattern. The red squares (males) and
red circles (females) show how mtDNA is passed on from the mother to all her
children.
Testing Methodology
The discriminatory power of mtDNA testing arises from the polymorphic nature
(for unrelated individuals) of the two hypervariable regions HV1 and HV2 located
within the D-loop of the mtDNA. To determine these polymorphisms, the HV1
region (nucleotides 16010-16400) and the HV2 region (nucleotides 50-470) of
the mtDNA are sequenced. The sequence data of the test sample and maternal
reference are then compared to the standard sequence, the Cambridge
Reference Sequence. Any differences between the test sample and the maternal
56

eference sample from the standard sequence are recorded. A similar set of
differences of the test sample and maternal reference from the standard
sequence indicates relatedness.
Indications for mtDNA Sequence Analysis
• To determine if siblings (brothers and/or sisters) have the same mother
• To determine relatedness from mother’s side of the family (uncles, aunts,
grandmothers, etc.)
• To reconstruct family maternal-linked relationships
• In cases were DNA sample concentration is very low or degraded,
especially in forensic cases. (Mitochondrial DNA sequencing is the
preferred methodology in these particular samples due to a higher
success rate).
Source: www.synergeneprofiling.co
57

Y-Chromosome Analysis (Y-STR Haplotypes)
Y-chromosome haplotypes are used to uniquely identify men. Except for two
small regions in which pairing and exchange take place with the X chromosome,
the Y-chromosome is not subject to recombination and is passed on through the
male line of the family largely unchanged. This allows a common paternal link to
be traced in their ancestral lineage. By looking at Y-chromosome markers from
all of the males with the same paternal descent we would suspect that all related
males should share the same Y-chromosome markers. Therefore, identical Y
STR patterns indicate inclusion within a family, whereas different Y STR patterns
indicate familial exclusion.
The figure below shows how the Y-chromosome (black squares) is passed from
father to son exclusively.
Testing Methodology
Synergene utilises the PowerPlex Y STR kit (Promega), which allows analysis of
12 Y STR loci. Results from this analysis are compared to a population Y
haplotype database.
Indications for Y STR Analysis
• To determine if males are from the same paternal lineage
• To determine relatedness from father’s side of the family
• To reconstruct family paternal-linked relationships
• In sexual Assault cases, to distinguish the male sample from mixed
samples: Source: www.synergeneprofiling.com
58

III. Interesting Cases Using DNA Evidence
Snowball the Cat
The elusive cat—both revered and demonized throughout the course of human
history—has become one of the animals most important to helping scientists
understand human genetics. Marilyn Menotti-Raymond is among a group of
scientists studying and developing a map of the cat genome at the National
Cancer Institute’s internationally renowned Laboratory of Genomic Diversity
(LGD) in Frederick, Maryland. Part of the National Institutes of Health, it is the
only research laboratory in the world attempting this work. “We are a cat genome
center,” Menotti-Raymond says. “We have 15 researchers working on many
aspects of the cat genome, from constructing genetic maps of the cat to research
in natural populations of exotic felids.”
It turns out that cats and humans have much in common in terms of how their
genes are ordered and organized, Menotti-Raymond says. Cats have 19 pairs of
chromosomes, including one pair of sex chromosomes; humans have 23 pairs of
chromosomes, including one pair of sex chromosomes. “If you align human and
cat chromosomes, the gene order and organization are more alike than with any
other mammalian species whose genomes have been examined, except for
some of the primate species,” she says. Because of the similarities, scientists
believe it will be easier to identify hereditary diseases caused by defects in genes
that are analogous to both cats and humans. In doing so, researchers hope to
establish the cat as a useful model to further the understanding of some 200
human hereditary diseases; tumorous conditions called neoplasia; genetic
factors related to infectious diseases; and mammalian genome evolution.
At LGD, Menotti-Raymond is a staff scientist in the animal genetics group,
where she works with five other researchers and several graduate students and
postdoctoral fellows. “The work can sometimes be frustrating,” she says. “You
have to like the process and be satisfied with the pursuit of knowledge.”
Menotti-Raymond’s career took an unusual turn when a cat became a key
part of a murder case on Prince Edward Island, Canada. In 1994, Shirley
Duguay, a 32-year-old mother of five, disappeared. Her body was found in a
shallow grave a few months later. Among the chief suspects in the murder was
the woman’s estranged common-law husband, Douglas Beamish, who was living
nearby in his parents’ home. Royal Canadian Mounted Police had no evidence
linking Beamish to the crime. During the search for the victim’s body, however,
the Mounties discovered a plastic bag containing a leather jacket with blood
stains that matched the victim’s blood. The jacket also contained 27 strands of
white hair, which forensic investigators determined were from a cat. The
Mounties remembered a white cat named Snowball living in Beamish’s parents’
home. The trick was to prove the cat hair found in the jacket was Snowball’s. A
Mountie investigator used the Internet to search for an expert in cat genomes,
which led him to Menotti-Raymond and LGD director Stephen J. O’Brien. “They
wanted to know if we could do a DNA fingerprint of the cat hair,” Menotti-
Raymond says. “We had the genetic tools to do it, but it became a question of
59

whether we wanted to get involved in forensics, and whether we could isolate
enough DNA from a single hair specimen to perform the analysis. We decided to
proceed and determined there was a match between the cat and the hair found
in the jacket.”
Menotti-Raymond and O’Brien became expert witnesses during the murder
trial, and their evidence helped convict Beamish. The case set a legal precedent
as the first to allow animal DNA-typing data as evidence in a court proceeding.
After-ward, the lab received numerous requests from across the United States for
similar DNA typing. But LGD researchers simply did not have the time,
resources, or mandate from the NIH to devote to forensic investigations on a
large scale. The solution: Develop tools so that other facilities could do this kind
of forensic work. Last year, Menotti-Raymond received a $265,000 grant from the
U.S. Department of Justice to develop the National Feline Genetic Database—
the first of its kind. The researchers are using the grant to develop molecular
tools that will enable forensic laboratories to do DNA analyses on cat
specimens—hair, blood, or tissue samples. “The goal is to develop the molecular
tools needed to characterize cat specimens left at crime scenes and to create a
genetic database that can be used to evaluate matching profiles,” Menotti-
Raymond says.
To develop the database, Menotti-Raymond’s research group is trying to
collect about 50 specimens from each breed of cat. There are about 35 different
breeds, which means some 1,750 samples are needed. To drum up interest in
the project, Menotti-Raymond sent out mass mailings to cat breeders and has
visited numerous cat shows. So far, the lab has collected more than 850
samples.
Menotti-Raymond began working at LGD after completing a Ph.D. in
molecular biology at SU in 1990. Much like the fabled nine lives of a cat, the
Fayetteville, New York, native has undergone a few incarnations of her own.
Although she always wanted to study microbiology like her father, Amel Menotti,
a chemist and the first director of research at Bristol Laboratories in Syracuse,
her life took a detour at Denison University in Ohio. During her sophomore year
there, she married and returned to the Syracuse area with her husband.
She earned a bachelor’s degree in bacteriology at Syracuse University in
1966, and, after having two sons, James and Daniel, completed a master’s
degree in science teaching in 1971. When the boys reached high-school age,
Menotti-Raymond returned to college to pursue her dream of becoming a
biologist. “I felt I had left something undone,” she says. “I initially went back to
college to complete a master’s degree in biology and then decided to continue on
in the Ph.D. program.”
Menotti-Raymond studied with College of Arts and Sciences biology
professor David Sullivan, and, as a doctoral student, researched genetic
regulation in drosophila (fruit flies). The work resulted in the publication of three
papers that she co-authored with Sullivan, who suggested she interview for a
position at LGD after she graduated. Since then, she has authored or coauthored
more than 20 articles in her field. “I am fortunate to have spent the early
years of my children’s lives at home with them,” she says. “That was important to
60

me. But I was also lucky that when I decided to return to school, it was only
seven miles to a university where I found an excellent mentor and an excellent
laboratory.”
Source:
http://sumagazine.syr.edu/summer01/features/brightideas/brightpg2.html
61

DNA & O.J.
by Katherine Ramsland
A barking dog alerted a neighbor to the crime scene. Sukru Boztepe followed the dog
back to the Brentwood condominium, saw the horrendous bloodshed, and urged his wife
to phone 911. That set into motion the initial events in a convoluted series that made up
what many called "the Crime of the Century." It also brought DNA testing in criminal
cases to public awareness.
Nicole Brown Simpson, former wife of former football celebrity
O. J. Simpson, went outside her home late in the evening of
June 12, 1994, and was met by an assailant who slashed her to
death. The killer also slaughtered the man who was with her,
Ronald Goldman, age 25. He had brought Nicole the
eyeglasses that her mother had left behind at the restaurant
where he was a waiter. They were both found dead, covered in
blood, just inside the front gate.
O.J. Simpson booking
photo (AP)
62
Ronald Goldman & Nicole Simp
Although Nicole was no longer married to Simpson, the police
contacted him right away. Going to his home, detectives noted a
bloodstain on the door of his white Ford Bronco. A trail of blood also
led up to the house, but Simpson appeared to be gone. It turned out
that he had just flown to Chicago.
He returned to Los Angeles and agreed to answer questions. Investigators then noticed
a cut on a finger of his left hand that would prove to be problematic for him when they
eventually charged him with the crimes. First, he told several conflicting stories about
how he had gotten the cut, and second, the crime scene indicated that the killer had
been cut on his left hand and had trailed blood outside the gates. That hardly seemed
coincidental. Nevertheless, another narrative eventually overshadowed these problems.
Several droplets of blood at the scene failed to show a match with either of the victim's
blood types. Then Simpson's blood was drawn for testing (after the droplets had already
been collected) and comparison between Simpson's DNA and that of the blood at the
scene showed strong similarities. Contrary to what Simpson's defense team was to say
after his arrest, this blood could not have been planted after Simpson's blood was
drawn.

The tests indicated that the drops had three factors in common with Simpson's blood
and only one person in 57 billion could produce an equivalent match. In addition, the
blood was found near footprints made by a rare and expensive type of shoe—shoes that
O. J. wore and that proved to be his size.
Next to the bodies was a bloodstained black leather glove that bore traces of fiber from
Goldman's jeans. The glove's mate, stained with Simpson's blood, was found on his
property. There were also traces of the blood of both victims lifted from inside
Simpson's car and house, along with blood that contained his own DNA. In fact, his
blood and Goldman's were found together on the car's console.
Forensic serologists at the California Department of Justice, along with a private
contractor, did the sophisticated DNA testing. Then other evidence emerged, such as
the testimony of the limousine driver who came to pick Simpson up for the ride to the
airport: He saw a black man cross the driveway and go into the house. Then Simpson
claimed that the driver had been unable to get him on the intercom because he had
"overslept." There were also photos of Nicole and diary entries that attested to
Simpson's abusive and stalking behavior. In addition, when Simpson was notified that
he would be arrested for murder, he fled with his friend, Al Cowlings, and hinted in a
note that he might kill himself. With him were a passport, fake beard, and thousands of
dollars in cash. Nevertheless, he pleaded Not Guilty, offered a huge reward for
information about the crime, and hired a defense team of celebrity lawyers. Barry
Scheck and Peter Neufeld, from New York, were the DNA experts, renowned for their
work on the Innocence Project, which used DNA analysis to defend the falsely accused.
The defense team was going to call for a pretrial hearing on DNA evidence, to challenge
it from every angle, but decided instead to drop it. In part, they knew that whatever
happened could set a dangerous precedent and in part they realized that prolonging the
trial process could antagonize the jury. They wanted the jury on their side. So they
waived the proceeding, which many defense strategists felt was a radical decision, and
went on with the trial. Barry Scheck felt confident that they could produce challenges in
court before the jury that would accomplish all they wanted and also educate and
persuade the jury.
The reliability of this evidence came to be called the "DNA Wars," and three different
crime labs performed the analysis. All three determined that the DNA in the drops of
blood at the scene matched Simpson's. It was a 1 in 170 million match, using one type
of analysis known as RFLP, and 1 in 240 million match using the PCR test.
Nevertheless, criminologist Dr. Henry Lee testified that there appeared to be something
wrong with the way the blood was packaged, leading the defense to propose that the
multiple samples had been switched. They also claimed that the blood had been
severely degraded by being stored in a lab truck, but the prosecution's DNA expert,
Harlan Levy, said that the degradation would not have been sufficient to prevent
accurate DNA analysis. He also pointed out that control samples were used that would
have shown any such contamination, but Scheck suggested that the control samples
63

had been mishandled by the lab—all five of them.
What hurt the prosecution's case more than anything else were the endless explanations
of the complex procedures involved in DNA analysis. The defense kept it simple and
thereby befriended the jury. They then intimated that Detective Mark Fuhrman, who had
been at O. J.'s home the night of the murder, was a racist and had planted evidence.
They offered no proof of the latter statement, but allowed it to flow from the former,
which they did manage to prove.
The evidence was damning, but the defense team managed to refocus the jury's
attention on the corruption in the Los Angeles Police Department. Then Simpson made
a clear statement of his innocence, though he was not on the stand, and the defense
attorneys disputed the good reputation of the forensics labs, proving that the evidence
had been carelessly handled. Deliberating less than four hours, the jury bought all of
this and freed Simpson with a Not Guilty verdict. They defended themselves in
interviews after the fact by simply stating that the prosecution had not made its case. It
may be that those attorneys made some serious errors, but the doubt by the defense
about DNA was ludicrous and did some damage with the public to the credibility of this
type of evidence.
However, when it was first used in England as a way to determine the guilt of an
offender, it proved to be quite impressive.
Source: http://www.crimelibrary.com/criminal_mind/forensics/dna/1.html?sect=21
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July 17, 1918. Czar Nicholas II, his wife Empress Alexandra, their five children,
their doctor and three attendants are herded into the cellar of a house in
Yekaterinburg. Nicholas had been forced to abdicate a year earlier as civil war
gripped the country. He and his family were forced into exile the Ural Mountains
city. The Czar and his family were lined up against a wall, for a family portrait,
their Bolshevik captors said. Instead, a firing squad burst into the room and
opened fire. Jewels sewn into the dresses of the women deflected some of the
bullets. The firing squad used bayonets to finish them off.
The bodies were dumped into a mine shaft. They were later retrieved as word of
the killings spread. The death squad tried burning two of the bodies - but it took
too long. They doused the rest of the bodies with sulfuric acid and buried them in
a shallow grave in a forest outside the city.
In the late 1970's, a local geologist and a filmmaker began their quest to find the
site. They retrieved three skulls but replaced them and held onto their secret until
1989, as Mikhail Gorbachev's glasnost opened doors previously slammed shut.
In 1991, the bodies were exhumed - the same month that Boris Yeltsin became
Russia's first president.
DNA testing identifies Czar's remains
The process to identify the remains was exhaustive. Samples were sent to Britain
and the United States for DNA testing. The tests concluded that five of the
skeletons were members of one family and four were unrelated. Three of the five
were determined to be the children of two parents. The mother was linked to the
British royal family, as was Alexandra. The father was determined to be related to
several other Romanovs. Scientists said they were more than 99 percent sure
that the remains were those of the Czar, his family and their attendants. Two
skeletons remain unaccounted for - Alexei, the 13 year old heir to the throne, and
one of his sisters, either Maria or Anastasia.
Healing decades of excess
After the identities of the skeletons were established, President Boris Yeltsin
declared that the remains should receive a state funeral and be interred at the
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imperial capital of St. Petersburg, along with the remains of other Czars. It was to
be a three-day affair, presided over by Yeltsin and the Russian Orthodox
patriarch - and attended by ranks of royalty and world leaders. The skeletons
were to travel to St. Petersburg in a special train, making stops along the way.
It was seen as a way of putting to rest the past, a way to ask forgiveness for the
sins of communism. But instead of healing, the bones of the Czar have caused
even further division in a nation struggling to emerge from economic and political
turmoil.
Debate over the bones
Just three days before the scheduled burial, Russian patriarch Alexy II went on
national television to launch a virulent attack on the authenticity of the remains. In
a rare broadcast to the nation, the patriarch savaged an official government
commission which identified the remains as those of Russia's last Czar and his
family. Alexy II bitterly attacked the decision to go ahead with the funeral despite
doubts about the authenticity of the remains expressed "by experienced
scientists" on the commission. The patriarch said the area where the bones were
found was often used for mass executions during the civil war. The church
wanted the bones to be buried in a symbolic tomb until the last doubts about their
identity can be removed.
In Toronto, Olga Kulikovsky-Romanov, the widow of one of the Czar's nephews,
was one of the Romanov relatives who stayed away from the funeral. She, too,
disputes the authenticity of the remains.
"They couldn't even prove anything with O.J. Simpson's DNA, and here are
bones that have been dug up after 80 years, and they think they can prove it's
the same family," she told Reuters news agency.
Church rift
The debate over the authenticity of the bones may be little more than a wish by
the Orthodox church in Russia not to offend the Russian Orthodox church
abroad. The church in exile was founded by clerics and members who fled
Russia during the civil war. It canonized Czar Nicholas and his family in a
ceremony in New York in 1981. The church abroad considers the bones sacred
relics, which means they could not be placed in the Romanov family tomb.
The church abroad has made canonization of the Romanovs a precondition for
re-unification of the church, which would boost the Russian Orthodox Church's
position within Russia and among other branches of Orthodoxy.
The church within Russia is moving towards canonization, but it's expected to
take several years.
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Guest list pared
With the refusal of Patriarch Alexy II to attend, President Yeltsin said he would
not show up. Many say Yeltsin did not want to offend the church. In a scathing
editorial on Tueday, July 14, the Moscow Times chastized Yeltsin for refusing to
attend.
``It could have marked a closing of the books of Communism, an admission of
the old regime's excesses and a desire to look to the future,'' the newspaper said
in an editorial. ``But it now looks like it will turn into one of the most cowardly,
self-seeking and, frankly, stupid cat fights in the history of post-Soviet Russia.''
Just a day before the funeral, Yeltsin changed his mind, instantly elevating the
importance of the occasion.
Yeltsin said burial of Russia's last Czar and his family should redeem the sins of
the ancestors who killed them 80 years ago and tried to justify the murder
afterwards. ``The burial is an act of humane justice, a symbol of unification in
Russia and redemption of common guilt,'' Yeltsin told mourners in the former
imperial capital's Saint Peter and Paul Cathedral where the funeral took place.
``We must end the century which has been an age of blood and violence in
Russia with repentance and peace, regardless of political views, ethnic or
religious belonging,'' he said in a powerful, moving speech.
The diocese of St. Petersburg assigned seven local priests and and five deacons
to preside over the service. As the bones of the Czar were laid to rest, the
Romanov name was not even be mentioned. Instead, a prayer was said for all
the victims of oppression during the civil war and the years that followed.
Source: http://radio.cbc.ca/news/czar/
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A RAPE DEFENDANT WITH NO IDENTITY, BUT A DNA PROFILE
October 7, 1999 –
New York Times
By BILL DEDMAN
In an unusual legal strategy that stretches the law to match the developing
science of DNA, a prosecutor in Wisconsin has filed rape and kidnapping
charges against a defendant who has no name. John Doe is known only by his
DNA code, extracted from semen samples from three rapes and tested after six
years in a police property room.
''We know that one person raped these three women,'' said the prosecutor,
Norman Gahn, assistant district attorney for Milwaukee County. ''We just don't
know who that person is. We will catch him.''
The prosecutor's strategy is an effort to keep the six-year time limit for bringing
charges from running out. A warrant must identify a person to be arrested, and
this one names ''John Doe, unknown male with matching deoxyribonucleic acid
profile.'' Every month, an F.B.I. computer will compare the description of John
Doe's DNA with thousands of new genetic samples from around the country.
''John Doe'' is typically used in a warrant when the accused is known by an alias
or by a physical description. The Milwaukee case is unusual in that it describes
the suspect by only DNA information.
The case raises several questions about the gaps between science and law. Can
a person be identified on a warrant by only a DNA code, or is a more traditional
identification required, like a name or a physical description? Is the legal concept
of a statute of limitations relevant in an age of DNA testing? Legal experts said
the case had merit but would be challenged.
The case also reveals the promise and limitations of the national DNA databank,
one year old this month, which, much like a national fingerprint databank, was
intended to allow investigators to share in the DNA information collected by state
and municipal law-enforcement agencies.
The Federal Bureau of Investigation says the databank had helped solve 583
cases, including one involving a serial rapist in Washington, D.C., who turned out
to also be a serial rapist in Florida. Police routinely collect DNA evidence not only
in rape cases but also from saliva on beer cans left by a burglar, from sweat on a
baseball bat used in a beating and from blood on a bullet that passed through a
suspect.
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But only 18 of the states are hooked into the databank, the Combined DNA Index
System, or Codis, while the rest are working on the technology and the financing.
The databank is far smaller than the F.B.I.'s fingerprint bank. Much of the DNA
evidence that is collected never makes its way into the databank.
For example, about 180,000 boxes of evidence, known as rape kits, sit
unexamined on shelves in police departments across the nation, according to a
survey of police agencies for the National Commission for the Future of DNA
Evidence. These flat, rectangular boxes include blood vials, swabs of semen,
locks of hair and fingernail scrapings -- evidence collected from rape victims.
New York City alone has 12,000 unexamined rape kits and is trying to reduce the
backlog by contracting with private laboratories. It has stopped destroying
untested kits after five years, but other police departments continue the practice.
''We have the technology to solve these crimes, and we're not doing it,'' said
Christopher Asplen, executive director of the DNA commission, which
commissioned the survey.
Many police departments still use DNA evidence the way they have used
fingerprints and tire tracks, to determine whether a suspect committed the crime.
But the DNA databank makes the evidence useful for matching cases to
convicted offenders, or to other cases in which a suspect may be known.
Cities vary widely in the use of DNA testing.
In Cleveland, ''We only do DNA testing when there's an identifiable suspect,'' said
Lieut. Edward Thiery, a spokesman for the Police Department.
In Chicago, ''We now submit every sample for testing, but we still have a backlog
of untested samples,'' said Pat Camden, a Police Department spokesman.
In Los Angeles, the Police Department buys a 40-foot refrigerated trailer truck
every six months, just to hold DNA evidence.
''The Los Angeles Police Department, right now, if you didn't have a suspect in
custody and a court date, that thing is not going to get analyzed, whatever it is,''
Maria Foster, detective supervisor, told the DNA Commission.
The three DNA samples that uncovered a serial rapist in Milwaukee were
collected in 1993. In each case, a man grabbed a woman from behind, told her
that he had a knife, took her to a quiet place, raped her and then demanded
money, the police said. After each woman reported the crime, vaginal and
cervical swabs of semen were collected at a hospital.
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The samples sat in the police evidence room, along with 2,300 other unexamined
rape kits, for almost six years. To test them all would have swamped the state
crime laboratory.
So Detective Lori Gaglione and her colleagues in Milwaukee's Sensitive Crimes
Unit narrowed down the 2,300 cases to 53 based on such factors as the
availability of victims and witnesses.
From the 53 samples that were tested, one case matched a convicted rapist in
Minnesota. Another matched a convicted rapist elsewhere in Wisconsin, allowing
charges to be filed only eight hours before the statute of limitations expired. And
the three samples from 1993 matched each other. The other 47 remain in the
national databank.
The Wisconsin state law, as in other states, requires that a warrant identify the
accused. If the name is not known, the law allows identification ''by any
description by which the person to be arrested can be identified with reasonable
certainty.''
''My argument is going to be that genetic code goes well beyond reasonable
certainty,'' said Mr. Gahn, who is a member of the Federal DNA Commission.
''We're pushing the envelope as far as we can. It does something for the victim of
the sexual assault, to know that someone cares and to know that we're out there
working on the case.''
The forensic science supervisor in the Wisconsin State Crime Laboratory, Dirk
Janssen, said the probability of randomly selecting an unrelated individual who
would have a DNA profile matching the three samples would be about 1 in 7.25
billion -- more than the world's population -- in the United States Caucasian
population, and 1 in 1.96 billion in the African-American population.
Such a John Doe warrant based on DNA evidence has been used at least one
other time, in Kansas in 1991. No one was arrested, so the issue has not been
tested.
The warrant, which was signed by a judge last month, appeared to have merit,
said Myrna Raeder, a professor of law at Southwestern University in Los Angeles
and the former head of the criminal section of the American Bar Association.
''It's clearly novel and therefore courts are really going to have to struggle with
the intention of the statute and whether the clear meaning of the statute would
cover this,'' Professor Raeder said.
In Wisconsin, as in most states, only people convicted of sexual offenses are
required to give samples for DNA testing. The Legislature is close to expanding
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that to all felony offenses, as Alabama and Virginia have done. The Legislature is
also considering eliminating the statute of limitations in cases of rape.
Someday, prosecutors hope, a man will leave a cigarette behind at another rape.
Or cut himself on broken glass at a burglary. His saliva or blood will be tested, its
DNA catalogued and entered in the F.B.I. databank. And he will be charged with
raping and kidnapping a woman in Milwaukee on Nov. 9, 1993.
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