DNA Evidence Presentation
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15 Biological Stain Analysis: DNA
Court POOL/ZUMA Press/Newscom
LEARNING OBJECTIVES
After studying this chapter, you should be able to:
• List the A-B-O antigens and antibodies found in each of the four blood types: A, B, AB, and O.
• List and describe forensic tests used to characterize a stain as blood.
• List the laboratory tests necessary to characterize seminal stains.
• Explain how to properly preserve suspect blood and semen stains for laboratory examination.
• Contrast chromosomes and genes.
• Name the parts of a nucleotide and explain how they are linked together to form DNA.
• Understand the concept of base pairing as it relates to the double-helix structure of DNA.
• Explain the technology of polymerase chain reaction (PCR) and how it applies to forensic DNA typing
• Understand the DNA-typing technique known as short tandem repeats (STRs).
• Describe the difference between nuclear and mitochondrial DNA.
• Understand the use of computerized DNA databases in criminal investigation.
• List the necessary procedures for the proper preservation of biological evidence for laboratory DNA
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analysis.
O. J. SIMPSON: A MOUNTAIN OF EVIDENCE
On June 12, 1994, police who arrived at the home of Nicole Simpson viewed a horrific scene. The bodies of O. J.
Simpson’s estranged wife and her friend Ron Goldman were found on the path leading to the front door of Nicole’s
home. Both bodies were covered in blood and had received deep knife wounds. Nicole’s head was nearly severed from
her body. This was not a well-planned murder. A trail of blood led away from the murder scene. Blood was found in O.
J. Simpson’s Bronco. There were blood drops on O. J.’s driveway and in the foyer of his home. A blood-soaked sock
was located in O. J. Simpson’s bedroom, and a bloodstained glove rested on the ground outside his residence (see
accompanying photo).
As DNA was extracted and profiled from each bloodstained article, a picture emerged that seemed to irrefutably link
Simpson to the murders. A trail of DNA leaving the crime scene was consistent with O. J.’s profile, as was the DNA
found in Simpson’s home. Simpson’s DNA profile was found in the Bronco along with that of both victims. The glove
contained the DNA profiles of Nicole and Ron, and the sock had Nicole’s DNA profile. At trial, the defense team
valiantly fought back. Miscues in evidence collection were craftily exploited. The defense strategy was to paint a
picture of not only an incompetent investigation but one that was tinged with dishonest police planting evidence. The
strategy worked. O. J. Simpson was acquitted of murder.
In 1901, Karl Landsteiner announced one of the most significant discoveries of the twentieth century—the typing of
blood—a finding that earned him a Nobel Prize twenty-nine years later. For years physicians had attempted to
transfuse blood from one individual to another. Their efforts often ended in failure because the transfused blood tended
to coagulate, or clot, in the body of the recipient, causing instantaneous death. Landsteiner was the first to recognize
that all human blood was not the same; instead, he found that blood is distinguishable by its group, or type.
Out of Landsteiner’s work came the classification system that we call the A-B-O system . Now physicians have the key
for properly matching the blood of a donor to that of a recipient. Because one blood type cannot be mixed with a
different blood type without disastrous consequences, this discovery, of course, had important implications for blood
transfusion, and millions of lives have since been saved.
Meanwhile, Landsteiner’s findings opened a new field of research in the biological sciences. Others began to pursue
the identification of additional characteristics that could further differentiate blood. By 1937, the Rh factor in blood
had been demonstrated, and shortly thereafter, numerous blood factors or groups were discovered. More than one
hundred blood factors have been identified. However, the ones in the A-B-O system are still the most important for
properly matching a donor and recipient for a transfusion.
Until the early 1990s, forensic scientists focused on blood factors, such as A-B-O, as offering the best means for
linking blood to an individual. What made these factors so attractive was that, in theory, no two individuals, except for
identical twins, could be expected to have the same combination of blood factors. In other words, blood factors are
controlled genetically and have the potential of being a highly distinctive feature for personal identification. What
makes this observation so relevant is the great frequency of bloodstains at crime scenes, especially crimes of the most
serious nature: homicides, assaults, and sexual assaults. Consider, for example, a transfer of blood between the victim
and assailant during a struggle, that is, the transfer of a victim’s blood to the suspect’s garment, or vice versa. If the
criminalist could individualize human blood by identifying all of its known factors, the result would be strong
evidence for linking the suspect to the crime.
The advent of DNA technology has dramatically altered the approach of forensic scientists toward the
individualization of bloodstains and other biological evidence. The search for genetically controlled blood factors in
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bloodstains has been abandoned in favor of characterizing biological evidence by select regions of our
deoxyribonucleic acid (DNA) , which carries the body’s genetic information. As a result, the individuation of dried
blood and other biological evidence has become a reality and has significantly altered the role that crime laboratories
play in criminal investigations. In fact, the high sensitivity of DNA analysis and the resultant search for DNA evidence
has even altered the types of materials collected from crime scenes.
deoxyribonucleic acid (DNA)
The molecules that carry the body’s genetic information.
The Nature of Blood
The word blood refers to a highly complex mixture of cells, enzymes, proteins, and inorganic substances. The fluid
portion of blood is called plasma ; it is composed principally of water and accounts for 55 percent of blood content.
Suspended in the plasma are solid materials consisting chiefly of several types of cells: red blood cells (i.e.,
erythrocytes), white blood cells (i.e., leukocytes), and platelets. The solid portion of blood accounts for 45 percent of
its content. Blood clots when a protein in the plasma known as fibrin traps and enmeshes the red blood cells. If the
clotted material were removed from the blood, a pale yellowish liquid known as serum would be left.
plasma
The fluid portion of unclotte blood.
serum
The liquid that separates from the blood when a clot is formed.
Considering the complexity of blood, a full discussion of its function and chemistry would extend beyond the scope of
this text. Instead, this chapter concentrates on the components of blood that are directly pertinent to the forensic
aspects of blood identification: the red blood cells and the blood serum.
ANTIGENS AND ANTIBODIES
Red blood cells transport oxygen from the lungs to the body tissues and remove carbon dioxide from tissues by
transporting it back to the lungs, where it is exhaled. However, for reasons unrelated to the red blood cell’s transporting
mission, on the surface of each cell are millions of characteristic chemical structures called antigens . Antigens impart
specific characteristics to the red blood cells. Blood antigens are grouped into systems depending on their relationship
to one another. More than fifteen blood antigen systems have been identified to date; of these, the A-B-O and Rh
systems are the most important.
antigen
A substance, usually a protein, that stimulates the body to produce antibodies against it.
If an individual has type A blood, this simply means that each red blood cell has A antigens on its surface; similarly,
all type B individuals have B antigens, and the red blood cells of type AB individuals contain both A and B antigens.
Type O individuals have neither A nor B antigens on their cells. Hence, the presence or absence of A and B antigens
on the red blood cells determines a person’s blood type in the A-B-O system.
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Another important blood antigen has been named the Rh factor , or D antigen. Those people who have the D antigen
are said to be Rh positive ; those without this antigen are Rh negative . In routine blood banking, the presence or absence
of the three antigens—A, B, and D—must be tested to determine the compatibility of the donor and recipient.
Serum is important because it contains proteins known as antibodies . The fundamental principle of blood typing is
that, for every antigen, there exists a specific antibody. Each antibody symbol contains the prefix anti- , followed by the
name of the antigen for which it is specific. Hence, anti-A is specific only for the A antigen, anti-B for the B antigen,
and anti-D for the D antigen. The antibody-containing serum is referred to as the antiserum , meaning a serum that
reacts against something (i.e., antigens).
antibody
A protein in the blood serum that destroys or inactivates a specific antigen.
antiserum
Blood serum that contains specific antibodies.
An antibody reacts only with its specific antigen and no other. Thus, if serum containing anti-B is added to red blood
cells carrying the B antigen, the two will combine, causing the antibody to attach itself to the cell. Antibodies are
normally bivalent —that is, they have two reactive sites. This means that each antibody can simultaneously be attached
to antigens located on two different red blood cells. This creates a vast network of cross-linked cells usually seen in the
form of clumping, or agglutination (see Figure 15-1 ).
agglutination
The clumping together of red blood cells by the action of an antibody.
FIGURE 15-1 Agglutination of blood cells.
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Let’s look a little more closely at this phenomenon. In normal blood, shown in Figure 15-2(a) , antigens on red blood
cells and antibodies coexist without destroying each other because the antibodies present are not specific toward any of
the antigens. However, suppose a foreign serum added to the blood introduces a new antibody. This results in a
specific antigen–antibody reaction that immediately causes the red blood cells to link together, or agglutinate, as
shown in Figure 15-2(b) .
FIGURE 15-2 (a) A microscopic view of normal red blood cells (500x). (b)
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A microscopic view of agglutinated red blood cells (500x).
Evidently, nature has taken this situation into account, for when we examine the serum of type A blood, we find anti-B
but no anti-A. Similarly, type B blood contains only anti-A, type O blood has both anti-A and anti-B, and type AB
blood contains neither anti-A nor anti-B. The antigen and antibody components of normal blood are summarized in the
following table:
Blood Type Antigens on Red Blood Cells Antibodies in Serum
A A Anti-B
B B Anti-A
AB AB Neither anti-A nor anti-B
O Neither A nor B Both anti-A and anti-B
The reasons for the fatal consequences of mixing incompatible blood during a transfusion should now be quite
obvious. For example, the transfusion of type A blood into a type B patient will cause the natural anti-A in the blood of
the type B patient to react promptly with the incoming A antigens, resulting in agglutination. In addition, the incoming
anti-B of the donor will react with the B antigens of the patient.
Immunoassay Techniques
The concept of a specific antigen-antibody reaction is being applied in other areas unrelated to blood typing. Most
significant, similar reactions are being applied to the detection of drugs in blood and urine. Antibodies that react with
drugs do not exist naturally; however, they can be produced in animals such as rabbits by first combining the drug with
a protein and injecting this combination into the animal. This drug-protein complex acts as an antigen stimulating the
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animal to produce antibodies (see Figure 15-3 ). The recovered blood serum of the animal now contains antibodies that
are specific or nearly specific to the drug.
FIGURE 15-3 Stimulating production of drug antibodies.
Currently, each day, thousands of individuals are voluntarily being subjected to urinalysis tests for the presence of
commonly abused drugs. These individuals include military personnel, transportation industry employees, police and
corrections personnel, and candidates undergoing preemployment drug screening. Immunoassay testing for drugs has
proved quite suitable for handling the large volume of specimens that must be rapidly analyzed on a daily basis for
drug content. Testing laboratories have available to them a variety of commercially prepared sera that were developed
in animals injected with any one of a variety of drugs. Once a particular serum is added to a urine specimen, it’s
designed to interact with either opiates, cannabinoids, amphetamines, phencyclidine, barbiturates, methadone, or
another type of drug that might be present. A word of caution: Immunoassay is only presumptive in nature, and its
result must be confirmed by additional testing.
Quick Review
• An antibody reacts or agglutinates only with its specific antigen. The concept of specific antigen-antibody
reactions has been applied to techniques for the detection of commonly abused drugs in blood and urine.
• Every red blood cell contains either an A antigen, a B antigen, both antigens, or no antigen (this is called type
O). The type of antigen on one’s red blood cells determines one’s A-B-O blood type. Persons with type A blood
have A antigens on their red blood cells, those with type B blood have B antigens, those with type AB blood
have both antigens, and those with type O blood have no antigens on their red blood cells.
• To produce antibodies capable of reacting with drugs, a specific drug is combined with a protein, and this
combination is injected into an animal such as a rabbit. This drug-protein complex acts as an antigen, stimulating
the animal to produce antibodies. The recovered blood serum of the animal will now contain antibodies that are
specific or nearly specific to the drug.
Forensic Characterization of Bloodstains
The criminalist must answer the following questions when examining dried blood: (1) Is it blood? (2) From what
species did the blood originate? (3) If the blood is human, how closely can it be associated with a particular individual?
COLOR TESTS
The determination that a substance is blood is best made by means of a preliminary color test. For many years, the
most common test was the benzidine color test . However, because benzidine has been identified as a known
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carcinogen, its use has generally been discontinued, and the chemical phenolphthalein is usually substituted (this test is
also known as the Kastle-Meyer color test ).
Both the benzidine and Kastle-Meyer color tests are based on the observation that blood hemoglobin possesses
peroxidase-like activity. Peroxi-dases are enzymes that accelerate the oxidation of several classes of organic
compounds when combined with peroxides. For example, when a bloodstain, phenolphthalein reagent, and hydrogen
peroxide are mixed together, oxidation of the hemoglobin in the blood produces a deep pink color.
The Kastle-Meyer test is not a specific test for blood; some vegetable materials, for instance, may turn Kastle-Meyer
pink. These substances include potatoes and horseradish. However, such materials will probably not be encountered in
criminal situations, and thus, from a practical point of view, a positive Kastle-Meyer test is highly indicative of blood.
Field investigators also have found Hemastix strips a useful presumptive field test for blood. Designed as a urine
dipstick test for blood, the strip can be moistened with distilled water and placed in contact with a suspect bloodstain.
The appearance of a green color indicates the presence of blood.
WebExtra 15.1
See a Color Test for Blood www.mycrimekit.com
LUMINOL AND BLUESTAR
Another important presumptive identification test for blood is the luminol test.
1
Unlike the benzidine and
Kastle-Meyer tests, the reaction of luminol with blood produces light rather than color. After spraying luminol reagent
onto suspect items, agents darken the room; any bloodstains produce a faint blue glow, known as luminescence . Using
luminol, investigators can quickly screen large areas for bloodstains. A relatively new product, Bluestar
( www.bluestar-forensic.com ), is now available to be used in place of luminol. Bluestar is easy to mix in the field. Its
reaction with blood can be observed readily without having to create complete darkness.
The luminol and Bluestar tests are extremely sensitive—capable of detecting bloodstains diluted up to 100,000 times.
For this reason, spraying large areas such as carpets, walls, flooring, or the interior of a vehicle may reveal blood traces
or patterns that would have gone unnoticed under normal lighting conditions (see Figure 15-4 ). Luminol and Bluestar
will not interfere with any subsequent DNA testing.
2
FIGURE 15-4 (a) A section of a linoleum floor photographed under
normal light. This floor was located in the residence of a missing person.
(b) The same section of the floor shown in (a) after spraying with luminol.
A circular pattern was revealed. Investigators concluded that the circular
blood pattern was left by the bottom of a bucket used during cleanup of
the blood. A small clump of sponge, blood, and hair was found near where
this photograph was taken.
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Courtesy North Carolina State Bureau of Investigation
MICROCRYSTALLINE TESTS
The identification of blood can be made more specific if microcrystalline tests are performed on the material. Several
tests are available; the two most popular ones are the Takayama and Teichmann tests . Both depend on the addition of
specific chemicals to the blood to form characteristic crystals containing hemoglobin derivatives. Crystal tests are far
less sensitive than color tests for blood identification and are more susceptible to interference from contaminants that
may be present in the stain.
PRECIPITIN TEST
Once the stain has been characterized as blood, the serologist determines whether the blood is of human or animal
origin. The standard test for this is the precipitin test. Precipitin tests are based on the fact that when animals (usually
rabbits) are injected with human blood, antibodies form that react with the invading human blood to neutralize its
presence. The investigator can recover these antibodies by bleeding the animal and isolating the blood serum, which
contains antibodies that specifically react with human antigens. For this reason, the serum is known as human
antiserum . In the same manner, by injecting rabbits with the blood of other known animals, virtually any kind of
animal antiserum can be produced. Antiserums are commercially available for human blood and for the blood of a
variety of commonly encountered animals, such as dogs, cats, and deer.
Several techniques have been devised for performing precipitin tests on bloodstains. The classic method is to layer an
extract of the bloodstain on top of the human antiserum in a capillary tube. Human blood—or, for that matter, any
protein of human origin in the extract—reacts specifically with antibodies present in the antiserum, indicated by the
formation of a cloudy ring or band at the interface of the two liquids (see Figure 15-5 ).
FIGURE 15-5 The precipitin test.
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GEL DIFFUSION
Another precipitin method, called gel diffusion , takes advantage of the fact that antibodies and antigens diffuse or
move toward one another on a plate coated with a gel medium made from a natural polymer called agar. The extracted
bloodstain and the human antiserum are placed in separate holes opposite each other on the gel. If the blood is human,
a line of precipitation forms where the antigens and antibodies meet.
Similarly, the antigens and antibodies can be induced to move toward one another under the influence of an electrical
field. In the electrophoretic method , an electrical potential is applied to the gel medium; a specific antigen–antibody
reaction is denoted by a line of precipitation formed between the hole containing the blood extract and the hole
containing the human antiserum (see Figure 15-6 ).
The precipitin test is very sensitive and requires only a small amount of blood for testing. Human bloodstains that have
been dry for ten to fifteen years and longer may still give a positive precipitin reaction. Even extracts of tissue from
mummies four to five thousand years old have given positive reactions with this test. Furthermore, human bloodstains
diluted by washing in water and left with only a faint color may still yield a positive precipitin reaction (see Figure
15-7 ).
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Once it has been determined that the bloodstain is human, an effort must be made to associate the stain with or
disassociate the stain from a particular individual. Until the mid-1990s, routine characterization of bloodstains
included the determination of A-B-O types; however, the widespread use of DNA profiling, or typing, has relegated
this subject to one of historical interest only.
FIGURE 15-6 Gel diffusion.
FIGURE 15-7 Results of the precipitin test of dilutions of human serum up
to 1 in 4,096 against a human antiserum. A reaction is visible for blood
dilutions up to 1 in 256.
Courtesy Millipore Biomedica, Acton, MA
Quick Review
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• The criminalist must be prepared to answer the following questions when examining dried blood: (1) Is it
blood? (2) From what species did the blood originate? (3) If the blood is of human origin, how closely can it be
associated to a particular individual?
• The determination that a substance is blood is best made by means of a preliminary color test. A positive result
from the Kastle-Meyer color test is highly indicative of blood.
• The luminol and Bluestar tests are used to search out trace amounts of blood located at crime scenes.
• The precipitin test uses antisera, normally derived from rabbits that have been injected with the blood of a
known animal, to determine the species origin of a questioned bloodstain.
Forensic Characterization of Semen
Many cases encountered in a forensic laboratory involve sexual offenses, making it necessary to examine evidence for
the presence of seminal stains. The forensic examination of articles for seminal stains can be considered a two-step
process. First, before any tests can be conducted, the stain must be located. Considering the potential number and
soiled condition of outer garments, undergarments, and possibly bed clothing submitted for examination, this can be an
arduous task. Once located, stains must be subjected to tests that will prove their identity. A stain may even be tested
for the blood type of the individual from whom it originated.
TESTING FOR SEMINAL STAINS
Often seminal stains are visible on a fabric because they exhibit a stiff, crusty appearance. However, reliance on such
appearance for locating the stain is unreliable and is useful only when the stain is in an obvious area. If the fabric has
been washed or contains only minute quantities of semen, visual examination offers little chance of detecting the stain.
The best way to locate and at the same time characterize a seminal stain is to perform the acid phosphatase color test .
ACID PHOSPHATASE TEST
Acid phosphatase is an enzyme that is secreted by the prostate gland into seminal fluid. Its concentrations in seminal
fluid are up to four hundred times those found in any other body fluid. Its presence can easily be detected when it
comes into contact with an acidic solution of sodium alpha naphthylphosphate and Fast Blue B dye. Also,
4-methylumbelliferyl phosphate (MUP) will fluoresce (i.e., emit light) under UV light when it comes into contact with
acid phosphatase.
acid phosphatase
An enzyme found in high concentrations in semen.
The utility of the acid phosphatase test is apparent when it becomes necessary to search many garments or large pieces
of fabric for seminal stains. Simply moistening a filter paper with water and rubbing it lightly over the suspect area
transfers any acid phosphatase present to the filter paper. Placing a drop or two of the sodium alpha naphthylphosphate
and Fast Blue B solution on the paper produces a purple color that indicates the acid phosphatase enzyme. In this
manner, any fabric or surface can be systematically searched for seminal stains.
If it is necessary to search extremely large areas—for example, a bedsheet or carpet—the article can be tested in
sections, narrowing the location of the stain with each successive test. Alternatively, the garment can be pressed
against a suitably sized piece of moistened filter paper. The paper is then sprayed with MUP solution. Semen stains
appear as strongly fluorescent areas under UV light. A negative reaction can be interpreted as an absence of semen.
Although some vegetable and fruit juices (such as cauliflower and watermelon), fungi, contraceptive creams, and
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vaginal secretions give a positive response to the acid phosphatase test, none of these substances normally reacts with
the speed of seminal fluid. A reaction time of less than 30 seconds is considered a strong indication of semen.
MICROSCOPIC EXAMINATION OF SEMEN
Semen can be unequivocally identified by the presence of spermatozoa. When spermatozoa are located through a
microscopic examination, the stain is definitely identified as having been derived from semen. Spermatozoa are
slender, elongated structures 50 to 70 microns long, each with a head and a thin flagellate tail (see Figure 15-8 ). The
criminalist can normally locate them by immersing the stained material in a small volume of water. Rapidly stirring the
liquid transfers a small percentage of the spermatozoa present into the water. A drop of the water is dried onto a
microscope slide, then stained and examined under a compound microscope at a magnification of approximately 400×.
Considering the extremely large number of spermatozoa found in seminal fluid (the normal male releases 250 to 600
million spermatozoa during ejaculation), the chance of locating one should be very good; however, this is not always
true. One reason is that spermatozoa bind tightly to cloth materials.
3
Also, spermatozoa are extremely brittle when dry
and easily disintegrate if the stain is washed or when the stain is rubbed against another object, as happens frequently
in the handling and packaging of this type of evidence. Furthermore, sexual crimes may involve males who have an
abnormally low sperm count, a condition known as oligospermia , or who have no spermatozoa at all in their seminal
fluid ( aspermia ). Significantly, aspermatic individuals are increasing in numbers because of the growing popularity of
vasectomies.
oligospermia
An abnormally low sperm count.
aspermia
The absence of sperm; sterility in males.
FIGURE 15-8 A photomicrograph of human spermatozoa (300×).
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John Walsh\Photo Researchers, Inc.
PROSTATE-SPECIFIC ANTIGEN (PSA)
Analysts often examine stains or swabs that they suspect contain semen (because of the presence of acid phosphatase),
but that yield no detectable spermatozoa. How, then, can one reliably prove the presence of semen? The solution to this
problem came with the discovery in the 1970s of a protein called p30 or prostate-specific antigen (PSA) . At first, this
protein was thought to be prostate specific and hence a unique identifier of semen. However, additional research has
shown that low levels of p30 may be detectable in other human tissues. A more reasonable approach to the
unequivocal identification of semen is to use a positive p30 test in combination with an acid phosphatase color test
with a reaction time of less than 30 seconds.
4
When p30 is isolated and injected into a rabbit, it stimulates the production of polyclonal antibodies (anti-p30). The
serum collected from these immunized rabbits can then be used to test suspected semen stains. As shown in Figure
15-9 , the stain extract is placed in one well of an electrophoretic plate and the anti-p30 in an opposite well. When an
electric potential is applied, the antigens and antibodies move toward each other. The formation of a visible line
midway between the two wells shows the presence of p30 in the stain and indicates that the stain originated from
semen.
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FIGURE 15-9 PSA testing by electrophoresis.
FIGURE 15-10 An antibody–antigen–antibody “sandwich,” or complex, is
seen as a colored band arising from the attached blue dye. This signifies
the presence of PSA in the extract of a stain and positively identifies
human semen.
A more elegant approach to identifying PSA (or p30) is shown in Figure 15-10 . First, a monoclonal PSA antibody is
attached to a dye and placed on a porous membrane. Monoclonal antibodies are specially designed to attack a single
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antigen site. Next, an extract from a sample suspected of containing PSA is placed on the membrane. If PSA is present
in the extract, it combines with the monoclonal PSA antibody to form a PSA antigen–monoclonal PSA antibody
complex. This complex migrates along the membrane, where it interacts with a PSA antibody imbedded in the
membrane. The antibody–antigen–antibody “sandwich” that forms is apparent by the presence of a colored line (see
Figure 15-10 ). This monoclonal antibody technique is about 100 times as sensitive as the electrophoretic method for
detecting PSA.
Once the material is proved to be semen, the next task is to associate the semen as closely as possible with an
individual. As we will learn, forensic scientists can link seminal material to one individual with DNA technology. Just
as important is the fact that this technology can exonerate many of those wrongfully accused of sexual assault.
Quick Review
• The best way to locate and characterize a seminal stain is to perform the acid phosphatase color test.
• The presence of spermatozoa is a unique identifier of semen. Also, the protein called prostate-specific antigen
(PSA), also known as p30 , is useful in combination with the acid phosphatase color test for characterizing a
sample stain as semen.
• Forensic scientists can link seminal material to an individual by DNA typing.
Collection of Sexual Assault Evidence
Seminal constituents on a sexual assault victim are important evidence that sexual intercourse has taken place, but their
absence does not necessarily mean that a sexual assault did not occur. Physical injuries such as bruises and bleeding
tend to confirm that a violent assault occurred. Furthermore, the forceful physical contact between victim and assailant
may result in a transfer of physical evidence such as blood, semen, hairs, and fibers. The presence of such evidence
helps forge a vital link in the chain of circumstances surrounding a sexual crime.
To protect this kind of evidence, all the outer garments and undergarments from the victim should be carefully
removed and packaged separately in paper (not plastic) bags. A clean bedsheet should be placed on the floor and a
clean paper sheet placed over it. The victim must remove her shoes before standing on the paper. The person should
disrobe while standing on the paper in order to collect any loose foreign material falling from the clothing. Each piece
of clothing should be collected as it is removed and placed in a separate paper bag to avoid cross-contamination. The
paper sheet should be folded carefully so that all foreign materials are contained inside. If appropriate, bedding or the
object on which the assault took place should be submitted to the laboratory for processing.
Items suspected of containing seminal stains must be handled carefully. Folding an article at the location of a stain may
cause it to flake off, as will rubbing the stained area against the surface of the packaging material. If, under unusual
circumstances, it is not possible to transport the stained article to the laboratory, the stained area should be cut out and
submitted along with a separately packaged unstained piece as a substrate control.
In the laboratory, analysts try to link seminal material to a source using DNA typing. Because an investigator may
transfer his or her DNA types to a stain through perspiration, stained articles must be handled with care, minimizing
direct personal contact. The evidence collector must wear disposable latex gloves when such evidence must be
touched.
The sexual assault victim must undergo a medical examination as soon as possible after the assault. At this time, the
appropriate items of physical evidence are collected by trained personnel. Evidence collectors should have an
evidence-collection kit from the local crime laboratory (see Figure 15-11 ).
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The following procedure should be followed by a medical professional to collect items of physical evidence from the
sexual assault victim:
1. Pubic combings. Place a paper towel under the buttocks and comb the pubic area for loose or foreign hairs.
2. Pubic hair standard/reference samples. Cut fifteen to twenty full-length hairs from the pubic area at the skin
line.
3. External genital dry-skin areas. Swab with at least one dry swab and one moistening swab.
4. Vaginal swabs and smear. Using two swabs simultaneously, carefully swab the vaginal area and let the swabs
air-dry before packaging. Using two additional swabs, repeat the swabbing procedure and smear the swabs onto
separate microscope slides, allowing them to air-dry before packaging.
FIGURE 15-11 (left) A victim sexual assault evidence collection kit
showing the kit envelope, kit instructions, medical history and assault
information forms, and a foreign materials collection bag.
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FIGURE 15-11 (right) A victim sexual assault evidence collection kit
showing collection bags for outer clothing, underpants, debris, pubic
hair combings, pubic hair standard/reference samples, vaginal swabs,
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and rectal swabs.
Courtesy Tri-Tech, Inc., Southport, NC, www.tritechusa.com
FIGURE 15-11 A victim sexual assault evidence collection kit showing
collection bags for oral swabs and smear, standard/reference head
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hairs, saliva sample, and blood samples, and anatomical drawings.
Courtesy Tri-Tech, Inc., Southport, NC, www.tritechusa.com
5. Cervix swabs. Using two swabs simultaneously, carefully swab the cervix area and let the swabs air-dry
before packaging.
6. Rectal swabs and smear. To be taken when warranted by case history. Using two swabs simultaneously, swab
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the rectal canal, smearing one of the swabs onto a microscope slide. Allow both samples to air-dry before
packaging.
7. Oral swabs and smear. To be taken if oral–genital contact occurred. Use two swabs simultaneously to swab
the cheek area and gum line. Using both swabs, prepare one smear slide. Allow both swabs and the smear to
air-dry before packaging.
8. Head hairs. Cut at the skin line a minimum of ten full-length hairs from each of the following scalp locations:
center, front, back, left side, and right side. A total of at least fifty hairs should be cut and submitted to the
laboratory.
9. Blood sample. Collect at least 7 milliliters in a vacuum tube containing the preservative EDTA. (The blood
sample can be used for DNA typing as well as for toxicological analysis if required.)
10. Fingernail scrapings. Scrape the undersurface of the nails with a dull object over a piece of clean paper to
collect debris. Use separate paper, one for each hand.
11. All clothing. Package as described earlier.
12. Urine specimen. Collect 30 milliliters or more of urine from the victim for analysis for Rohypnol, GHB, and
other substances associated with drug-facilitated sexual assaults.
Often, during the investigation of a sexual assault, the victim reports that a perpetrator engaged in biting, sucking, or
licking areas of the victim’s body. As we will learn in the next section, the tremendous sensitivity associated with DNA
technology offers investigators the opportunity to identify a perpetrator DNA types from saliva residues collected off
the skin. The most efficient way to recover saliva residues from the skin is to first swab the suspect area with a rotating
motion using a cotton swab moistened with distilled water. A second, dry swab is then rotated over the skin to recover
the moist remains on the skin’s surface from the wet swab. The swabs are air-dried and packaged together as a single
sample.
If a suspect is apprehended, the following items are routinely collected:
1. All clothing and any other items believed to have been worn at the time of assault.
2. Pubic hair combings.
3. Head and pubic hair standard/reference samples .
4. A penile swab taken within 24 hours of the assault, when appropriate to the case history.
5. A blood sample or buccal swab for DNA typing purposes.
The advent of DNA profiling has forced investigators to rethink what items are evidential in a sexual assault. DNA
levels in the range of one-billionth of a gram are now routinely characterized in crime laboratories. In the past, scant
attention was paid to the underwear recovered from a male who was suspected of being involved in a sexual assault;
seminal constituents on a man’s underwear had little or no investigative value. Today, the sensitivity of DNA analysis
has created new areas of investigation. It is possible to link a victim and an assailant by analyzing biological material
recovered from the interior front surface of a male suspect’s underwear. This is especially important when
investigations have failed to yield the presence of the suspect’s DNA on evidence recovered from the victim.
CASE FILES
A common mode of DNA transfer occurs when skin cells from the walls of a female victim’s vagina are transferred
onto the suspect during intercourse. Subsequent penile contact with the inner surface of the suspect’s underwear often
leads to the recovery of the female victim’s DNA from the underwear’s inner surface. The power of DNA is illustrated
by a case in which the female victim of a sexual assault had consensual sexual intercourse with a male partner before
being assaulted by a different male. DNA extracted from the inside front area of the suspect’s underwear revealed a
female DNA profile matching that of the victim. The added bonus to investigators in this case was finding male DNA
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on the same underwear that matched that of the consensual partner.
Source: Based on information contained in Gary G. Verret, “Sexual Assault Cases with No Primary Transfer of Biological Material from Suspect
to Victim: Evidence of Secondary and Tertiary Transfer of Biological Material from Victim to Suspect’s Undergarments,” Proceedings of the
Canadian Society of Forensic Science , Toronto, Ontario, November 2001.
The persistence of seminal constituents in the vagina may help determine the time of an alleged sexual attack.
Although spermatozoa in the vaginal cavity provide evidence of intercourse, important information regarding the time
of sexual activity can be obtained from the knowledge that motile (living) sperm generally survive for up to six hours
in the vaginal cavity of a living female. However, a successful search for motile sperm requires a microscopic
examination of a vaginal smear immediately after it is taken from the victim.
A more extensive examination of vaginal collections is later made at a forensic laboratory. Nonmotile sperm may be
found in a living female for up to three days after intercourse and occasionally up to six days later. Intact sperm (i.e.,
sperm with tails) are not normally found more than 16 hours after intercourse, but they have been found as late as 72
hours later. The likelihood of finding seminal acid phosphatase in the vaginal cavity markedly decreases with time
following intercourse, with little chance of identifying this substance 48 hours after intercourse.
4
Hence, with the
possibility of prolonged persistence of both spermatozoa and acid phosphatase in the vaginal cavity after intercourse,
investigators should determine if and when voluntary sexual activity last occurred before the sexual assault. This
information will help in evaluating the significance of finding these seminal constituents in a female victim. Blood or
buccal swabs for DNA analysis should be taken from any consensual partner who had sex with the victim within 72
hours of the assault.
Another significant indicator of recent sexual activity is PSA. This semen marker normally is not detected in the
vaginal cavity beyond 72 hours following intercourse.
4
Quick Review
• A sexual assault victim should undergo a medical examination as soon as possible after the assault. At that
time clothing, hairs, and vaginal and rectal swabs can be collected for subsequent laboratory examination.
• The persistence of seminal constituents in the vagina may help determine the time of an alleged sexual attack.
Understanding DNA
The discovery of deoxyribonucleic acid (DNA), the deciphering of its structure, and the decoding of its genetic
information were turning points in our understanding of the underlying concepts of inheritance. Now, with incredible
speed, as molecular biologists unravel the basic structure of genes, we can create new products through genetic
engineering and develop diagnostic tools and treatments for genetic disorders.
For a number of years, these developments were of seemingly peripheral interest to forensic scientists. All that
changed when, in 1985, what started out as a more or less routine investigation into the structure of a human gene led
to the discovery that portions of the DNA structure of certain genes are as unique to each individual as fingerprints.
Alec Jeffreys and his colleagues at Leicester University, England, who were responsible for these revelations, named
the process for isolating and reading these DNA markers DNA fingerprinting . As researchers uncovered new
approaches and variations to the original Jeffreys technique, the terms DNA profiling and DNA typing came to be
applied to describe this relatively new technology.
This discovery caught the imagination of the forensic science community because forensic scientists have long
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searched for ways to definitively link biological evidence such as blood, semen, hair, and tissue to a single individual.
Although conventional testing procedures had gone a long way toward narrowing the source of biological materials,
individualization remained an elusive goal. DNA typing has allowed forensic scientists to accomplish this goal.
Although the technique is still relatively new, DNA typing has become routine in public crime laboratories. It also has
been made available to interested parties through the services of a number of skilled private laboratories. In the United
States, courts have overwhelmingly admitted DNA evidence and accepted the reliability of its scientific underpinnings.
GENES AND CHROMOSOMES
Hereditary material is transmitted via microscopic units called genes . The gene is the basic unit of heredity. Each gene
by itself or in concert with other genes controls the development of a specific characteristic in the new individual; the
genes determine the nature and growth of virtually every body structure.
gene
The basic unit of heredity, consisting of a DNA segment located on a chromosome.
The genes are positioned on chromosomes , threadlike bodies that appear in the nucleus of every body cell (see Figure
15-12 ). Almost all human cells contain forty-six chromosomes, mated in twenty-three pairs. The only exceptions are
the human reproductive cells, the egg and sperm , which contain twenty-three unmated chromosomes. During
fertilization, a sperm and egg combine so that each contributes twenty-three chromosomes to form the new cell
( zygote ). Hence, the new individual begins life properly, with twenty-three mated chromosome pairs. Because the
genes are positioned on the chromosomes, the new individual inherits genetic material from each parent.
chromosome
A threadlike structure in the cell nucleus composed of DNA, along which the genes are located.
egg
The female reproductive cell.
sperm
The male reproductive cell.
zygote
The cell arising from the union of an egg and a sperm cell.
FIGURE 15-12 A computer-enhanced photomicrograph image of human
chromosomes.
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Alfred Pasieka, Science Photo Library\Photo Researchers, Inc.
Actually, two dissimilar chromosomes are involved in the determination of sex. The egg cell always contains a long
chromosome known as the X chromosome ; the sperm cell may contain either a long X chromosome or a short Y
chromosome . When an X-carrying sperm fertilizes an egg, the new cell has two X chromosomes (i.e., XX) and
develops into a female. A Y-carrying sperm produces an XY fertilized egg and develops into a male. Because the
sperm cell determines the nature of the chromosome pair, we can say that the father biologically determines the sex of
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the child.
X chromosome
The female sex chromosome.
Y chromosome
The male sex chromosome.
ALLELES
Just as chromosomes come together in pairs, so do the genes they bear. The position a gene occupies on a chromosome
is its locus . Genes that govern a given characteristic are similarly positioned on the chromosomes inherited from the
mother and father. Thus, a gene for eye color on the mother’s chromosome will be aligned with a gene for eye color on
the corresponding chromosome inherited from the father. Alternative forms of genes that influence a given
characteristic and are aligned with one another on a chromosome pair are known as alleles .
locus
The physical location of a gene on a chromosome.
allele
Any of several alternative forms of a gene located at the same point on a particular pair of chromosomes.
Inheritance of blood type offers a simple example of allele genes in humans. An individual’s blood type is determined
by three genes, designated A, B, and O. A gene pair made up of two similar alleles—for example, AA and BB—is said
to be homozygous . For example, if the chromosome inherited from the father carries the A gene and the chromosome
inherited from the mother carries the same gene, the offspring will have an AA combination. Thus, when an individual
inherits two similar genes from his or her parents, there is no problem in determining the blood type of that person. An
individual with an AA combination will always be type A, a BB will be type B, and an OO will be type O.
homozygous
Having two identical allelic genes on two corresponding positions on a pair of chromosomes.
A gene pair made up of two different alleles—AO, for example—is said to be heterozygous . For example, if the
chromosome from one parent carries the A gene and the chromosome from the other parent carries the O gene, the
genetic makeup of the offspring will be AO. When two different genes are inherited, one gene will be dominant —that
is, the characteristic coded for by that gene is expressed. The other gene will be recessive —that is, its characteristics
remain hidden. In the case of blood types, A and B genes are dominant, and the O gene is recessive. Thus, with an AO
combination, A is always dominant over O, and the individual is typed as A. Similarly, a BO combination is typed as
B. In the case of AB, the genes are codominant , and the individual’s blood type will be AB. The recessive
characteristics of O appear only when both recessive genes are present in combination OO, which is typed simply as
O.
heterozygous
Having two different allelic genes on two corresponding positions on a pair of chromosomes.
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WebExtra 15.2
Learn About the Chromosomes as Present in Our Cells www.mycrimekit.com
WebExtra 15.3
Learn About the Structure of Our Genes www.mycrimekit.com
WebExtra 15.4
See How Genes Position Themselves on a Chromosome Pair www.mycrimekit.com
WebExtra 15.5
See How Genes Define Our Genetic Makeup www.mycrimekit.com
Quick Review
• The gene is the basic unit of heredity. A chromosome is a threadlike structure in the cell nucleus along which
the genes are located.
• Most human cells contain forty-six chromosomes, arranged in twenty-three mated pairs. The only exceptions
are the human reproductive cells, the egg and sperm, which contain twenty-three unmated chromosomes each.
• During fertilization, a sperm and an egg combine so that each contributes twenty-three chromosomes to form
the new cell, or zygote , that develops into the offspring.
• An allele is any of several alternative forms of genes that influence a given characteristic and that are aligned
with one another on a chromosome pair.
• A heterozygous gene pair is made up of two different alleles; a homozygous gene pair is made up of two
similar alleles.
• When two different genes are inherited, the characteristic in the dominant gene’s code will be expressed. The
characteristic in the recessive gene’s code will remain hidden.
WHAT IS DNA?
Inside each of 60 trillion cells in the human body are strands of genetic material called chromosomes. Arranged along
the chromosomes, like beads on a thread, are nearly 25,000 genes. The gene is the fundamental unit of heredity. It
instructs the body’s cells to make proteins that determine everything from hair color to susceptibility to diseases. Each
gene is composed of DNA designed to carry out a single body function.
Although DNA was first discovered in 1868, scientists were slow to understand and appreciate its fundamental role in
inheritance. Painstakingly, researchers developed evidence that DNA was probably the substance by which genetic
instructions are passed from one generation to the next. However, the first major breakthrough in comprehending how
DNA works did not occur until the early 1950s, when two researchers, James Watson and Francis Crick, deduced the
structure of DNA. It turns out that DNA is an extraordinary molecule skillfully designed to control the genetic traits of
all living cells, plant and animal.
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STRUCTURE OF DNA
Before examining the implications of Watson and Crick’s discovery, let’s see how DNA is constructed. DNA is a
polymer. A polymer is a very large molecule made by linking a series of repeating units, or monomers. In this case, the
units are known as nucleotides .
nucleotide
A repeating unit of DNA consisting of one of four bases—adenine, guanine, cytosine, or thymine—attached to a
phosphate-sugar group.
NUCLEOTIDES
A nucleotide is composed of a sugar molecule, a phosphorus atom surrounded by four oxygen atoms, and a nitrogen-
containing molecule called a base . Figure 15-13 shows how nucleotides can be strung together to form a DNA strand.
In this figure, S designates the sugar component, which is joined with a phosphate group to form the backbone of the
DNA strand. Projecting from the backbone are the bases.
The key to understanding how DNA works is to appreciate the fact that only four types of bases are associated with
DNA: adenine, cytosine, guanine, and thymine. To simplify our discussion of DNA, we will designate each of these
bases by the first letter of their names. Hence, A will stand for adenine, C for cytosine, G for guanine, and T for
thymine.
Again, notice in Figure 15-13 how the bases project from the backbone of DNA. Also, although this figure shows a
DNA strand of four bases, keep in mind that in theory there is no limit to the length of the DNA strand; a DNA strand
can be composed of a long chain with millions of bases. This information was well known to Watson and Crick by the
time they started detailing the structure of DNA. Their efforts led them to discover that the DNA molecule is
composed of two DNA strands coiled into a double helix . This can be thought of as resembling two wires twisted
around each other.
As Watson and Crick manipulated scale models of DNA strands, they realized that the only way the bases on each
strand could be properly aligned with each other in a double-helix configuration was to place base A opposite T and G
opposite C . Watson and Crick had solved the puzzle of the double helix and presented the world with a simple but
elegant picture of DNA (see Figure 15-14 ).
COMPLEMENTARY BASE PAIRING
The concept that the only arrangement possible in the double-helix configuration is the pairing of bases A to T and G to
C is known as complementary base pairing . Although A–T and G–C pairs are always required, there are no restrictions
on how the bases are sequenced on a DNA strand. Thus, one can observe the sequences T–A–T–T or G–T–A–A or
G–T–C–A . When these sequences are joined with their complements in a double-helix configuration, they pair as
follows:
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FIGURE 15-13 How nucleotides can be linked to form a DNA strand. S
designates the sugar component, which is joined with phosphate groups
( P ) to form the backbone of DNA. Projecting from the backbone are four
bases: A , adenine; G , guanine; T , thymine; and C , cytosine.
FIGURE 15-14 A representation of a DNA double helix. Notice how bases
G and C pair with each other, as do bases A and T . This is the only
arrangement in which two DNA strands can align with each other in a
double-helix configuration.
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Any base can follow another on a DNA strand, which means that the number of possible sequence combinations is
staggering. Consider that the average human chromosome has DNA containing 100 million base pairs. All of the
human chromosomes taken together contain about three billion base pairs. From these numbers, we can begin to
appreciate the diversity of DNA and, hence, the diversity of living organisms. DNA is like a book of instructions. The
alphabet used to create the book is simple enough: A, T, G , and C . The order in which these letters are arranged defines
the role and function of a DNA molecule.
Polymerase Chain Reaction (PCR)
Once the double-helix structure of DNA was discovered, how DNA duplicated itself prior to cell division became
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apparent. The concept of base pairing in DNA suggests the analogy of positive and negative photographic film. Each
strand of DNA in the double helix has the same information; one can make a positive print from a negative or a
negative from a positive.
WebExtra 15.6
What Is DNA? www.mycrimekit.com
PCR PROCESS
The synthesis of new DNA from existing DNA begins with the unwinding of the DNA strands in the double helix.
Each strand is then exposed to a collection of free nucleotides. Letter by letter, the double helix is re-created as the
nucleotides are assembled in the proper order, as dictated by the principle of base pairing ( A with T and G with C ). The
result is the emergence of two identical copies of DNA where before there was only one (see Figure 15-15 ). A cell can
now pass on its genetic identity when it divides.
Many enzymes and proteins are involved in unwinding the DNA strands, keeping the two DNA strands apart, and
assembling the new DNA strands. For example, DNA polymerases are enzymes that assemble a new DNA strand in
the proper base sequence determined by the original, or parent, DNA strand. DNA polymerases also “proofread” the
growing DNA double helices for mismatched base pairs, which are replaced with correct bases.
Until recently, the phenomenon of DNA replication appeared to be of only academic interest to forensic scientists
interested in DNA for identification. However, this changed when researchers perfected the technology of using DNA
polymerases to copy a DNA strand located outside a living cell. This laboratory technique is known as polymerase
chain reaction (PCR) . Put simply, PCR is a technique designed to copy or multiply DNA strands.
polymerase chain reaction (PCR)
A technique for replicating or copying a portion of a DNA strand outside a living cell.
In PCR, small quantities of DNA or broken pieces of DNA found in crime-scene evidence can be copied with the aid
of a DNA polymerase. The copying process is highly temperature dependent and can be accomplished in an automated
fashion using a DNA thermal cycler (see Figure 15-16 ). Each cycle of the PCR technique results in a doubling of the
DNA, as shown in Figure 15-15 . Within a few hours, thirty cycles can multiply DNA a billionfold. Once DNA copies
are in hand, they can be analyzed by any of the methods of modern molecular biology. The ability to multiply small
bits of DNA opens new and exciting avenues for forensic scientists to explore. It means that sample size is no longer a
limitation in characterizing DNA recovered from crime-scene evidence.
FIGURE 15-15 Replication of DNA. The strands of the original DNA
molecule are separated, and two new strands are assembled.
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FIGURE 15-16 The DNA thermal cycler, an instrument that automates
the rapid and precise temperature changes required to copy a DNA
strand. Within a matter of hours, DNA can be multiplied a billionfold.
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Applied Biosystems
Quick Review
• The gene is the fundamental unit of heredity. Each gene is composed of DNA specifically designed to control
the genetic traits of our cells.
• DNA is constructed as a very large molecule made of a linked series of repeating units called nucleotides .
• Four types of bases are associated with the DNA structure: adenine (A) , guanine (G) , cytosine (C) , and thymine
(T) .
• The bases on each strand of DNA are aligned in a double-helix configuration so that adenine pairs with
thymine and guanine pairs with cytosine. This concept is known as complementary base pairing .
• The order in which the base pairs are arranged defines the role and function of a DNA molecule.
• DNA replication begins with the unwinding of the DNA strands in the double helix. The double helix is
re-created as the nucleotides are assembled in the proper order (A with T and G with C) . Two identical copies of
DNA emerge from the process.
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• PCR (polymerase chain reaction) is a technique for replicating, or copying, a portion of a DNA strand outside a
living cell.
CLOSER ANALYSIS POLYMERASE CHAIN REACTION
The most important feature of PCR is the knowledge that an enzyme called DNA polymerase can be directed to
synthesize a specific region of DNA. In a relatively straightforward manner, PCR can be used to repeatedly duplicate
or amplify a strand of DNA millions of times. As an example, let’s consider a segment of DNA that we want to
duplicate by PCR:
To perform PCR on this DNA segment, short sequences of DNA on each side of the region of interest must be
identified. In the example shown here, the short sequences are designated by boldface letters in the DNA segment.
These short DNA segments must be available in a pure form known as a primer if the PCR technique is going to work.
The first step in PCR is to heat the DNA strands to about 94°C. At this temperature, the double-stranded DNA
molecules separate completely:
The second step is to add the primers to the separated strands and allow the primers to combine, or hybridize, with the
strands by lowering the test-tube temperature to about 60°C.
The third step is to add the DNA polymerase and a mixture of free nucleotides (A, C, G, T) to the separated strands.
When the test tube is heated to 72°C, the polymerase enzyme directs the rebuilding of a double-stranded DNA
molecule, extending the primers by adding the appropriate bases, one at a time, resulting in the production of two
complete pairs of double-stranded DNA segments:
This completes the first cycle of the PCR technique, which results in a doubling of the number of DNA molecules
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from one to two. The cycle of heating, cooling, and strand rebuilding is then repeated, resulting in a further doubling of
the DNA molecules. On completion of the second cycle, four double-stranded DNA molecules have been created from
the original double-stranded DNA sample. Typically, twenty-eight to thirty-two cycles are carried out to yield more
than one billion copies of the original DNA molecule. Each cycle takes less than two minutes.
DNA Typing with Short Tandem Repeats
Geneticists have discovered that portions of the DNA molecule contain sequences of letters that are repeated numerous
times. In fact, more than 30 percent of the human genome is composed of repeating segments of DNA. These
repeating sequences, or tandem repeats , seem to act as filler or spacers between the coding regions of DNA. Although
these repeating segments do not seem to affect our outward appearance or control any other basic genetic function,
they are nevertheless part of our genetic makeup, inherited from our parents. The origin and significance of these
tandem repeats is a mystery, but to forensic scientists they offer a means of distinguishing one individual from another
through DNA typing.
WebExtra 15.7
Polymerase Chain Reaction www.mycrimekit.com
SHORT TANDEM REPEATS (STRs)
Currently, short tandem repeat (STR) analysis has emerged as the most successful and widely used DNA-profiling
procedure. STRs are locations (loci) on the chromosome that contain short sequence elements that repeat themselves
within the DNA molecule. They serve as helpful markers for identification because they are found in great abundance
throughout the human genome.
short tandem repeat (STR) A region of a DNA molecule that contains short segments of three to seven repeating
base pairs.
STRs normally consist of repeating sequences of three to seven bases; the entire strand of an STR is also very short,
less than 450 bases long. These strands are significantly shorter than those encountered in other DNA typing
procedures. This means that STRs are much less susceptible to degradation and are often recovered from bodies or
stains that have been subject to extreme decomposition. Also, because of their shortness, STRs are an ideal candidate
for multiplication by PCR, thus overcoming the limited-sample-size problem often associated with crime-scene
evidence. Only the equivalent of eighteen DNA-containing cells is needed to obtain a DNA profile. For instance, STR
profiles have been used to identify the origin of saliva residue on envelopes, stamps, soda cans, and cigarette butts.
To understand the utility of STRs in forensic science, let’s look at one commonly used STR known as TH01. This
DNA segment contains the repeating sequence A–A–T–G . Seven TH01 variants have been identified in the human
genome. These variants contain five to eleven repeats of A–A–T–G . Figure 15-17 illustrates two such TH01 variants,
one containing six repeats and the other containing eight repeats of A–A–T–G .
During a forensic examination, TH01 is extracted from biological materials and amplified by PCR as described earlier.
The ability to copy an STR means that extremely small amounts of the molecule can be detected and analyzed. Once
the STRs have been copied or amplified, they are separated by electrophoresis. Here, the STRs are forced to move
across a gel-coated plate under the influence of an electrical potential. Smaller DNA fragments move along the plate
faster than do larger DNA fragments. By examining the distance the STR has migrated on the electrophoretic plate,
one can determine the number of A–A–T–G repeats in the STR. Every person has two STR types for TH01, one
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inherited from each parent. Thus, for example, one may find in a semen stain TH01 with six repeats and eight repeats.
This combination of TH01 is found in approximately 3.5 percent of the population. It is important to understand that
all humans have the same type of repeats, but there is tremendous variation in the number of repeats each of us has.
FIGURE 15-17 Variants of the short tandem repeat TH01. The upper
DNA strand contains six repeats of the sequence A–A–T–G ; the lower
DNA strand contains eight repeats of the sequence A–A–T–G . This DNA
type is known as TH01 6,8.
When examining an STR DNA pattern, one merely needs to look for a match between band sets. For example, in
Figure 15-18 DNA extracted from a crime-scene stain matches the DNA recovered from one of three suspects. When
comparing only one STR, a limited number of people in a population would have the same STR fragment pattern as
the suspect. However, by using additional STRs, a high degree of discrimination or complete individualization can be
achieved.
FIGURE 15-18 A DNA profile pattern of a suspect and its match to
crime-scene DNA. From left to right, lane 1 is a DNA standard marker;
lane 2 is the crime-scene DNA; and lanes 3 to 5 are control samples from
suspects 1, 2, and 3, respectively. Crime-scene DNA matches suspect 2.
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WebExtra 15.8
See the Thirteen CODIS STRs and Their Chromosomal Positions www.mycrimekit.com
WebExtra 15.9
Calculate the Frequency of Occurrence of a DNA Profile www.mycrimekit.com
WebExtra 15.10
Understand the Operational Principles of Capillary Electrophoresis www.mycrimekit.com
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MULTIPLEXING
What makes STRs so attractive to forensic scientists is that hundreds of types of STRs are found in human genes. The
more STRs one can characterize, the smaller the percentage of the population from which these STRs can emanate.
This gives rise to the concept of multiplexing . Using PCR technology, one can simultaneously extract and amplify a
combination of different STRs.
multiplexing
A technique that simultaneously detects more than one DNA marker in a single analysis.
One STR system on the commercial market is the STR Blue Kit. This kit provides the necessary materials for
amplifying and detecting three STRs (a process called triplexing ): D3S1358, vWA, and FGA. The design of the system
ensures that the size of the STRs does not overlap, thereby allowing each marker to be viewed clearly on an
electrophoretic gel, as shown in Figure 15-19 . In the United States, the forensic science community has standardized
thirteen STRs for entry into a national database known as the Combined DNA Index System (CODIS).
When an STR is selected for analysis, not only must the identity and number of core repeats be defined, but the
sequence of bases flanking the repeats must also be known. This knowledge allows commercial manufacturers of STR
typing kits to prepare the correct primers to delineate the STR segment to be amplified by PCR. Figure 15-20
illustrates how appropriate primers are used to define the region of DNA to be amplified. Also, a mix of different
primers aimed at different STRs will be used to simultaneously amplify a multitude of STRs (i.e., to multiplex). In
fact, one STR kit on the commercial market can simultaneously make copies of fifteen different STRs (see Figure
15-21 ).
DNA TYPING WITH STRs
The thirteen CODIS STRs are listed in Table 15.1 along with their probabilities of identity. The probability of identity
is a measure of the likelihood that two individuals selected at random will have an identical STR type. The smaller the
value of this probability, the more discriminating the STR. A high degree of discrimination and even individualization
can be attained by analyzing a combination of STRs (multiplexing). Because STRs occur independent of each other,
the probability of biological evidence having a particular combination of STR types is determined by the product of
their frequency of occurrence in a population. This combination is referred to as the product rule (see p. 107 ). Hence,
the greater the number of STRs characterized, the smaller the frequency of occurrence of the analyzed sample in the
general population.
The combination of the first three STRs shown in Table 15.1 typically produces a frequency of occurrence of about 1
in 5,000. A combination of the first six STRs typically yields a frequency of occurrence in the range of 1 in 2 million
for the Caucasian population, and if the top nine STRs are determined in combination, this frequency declines to about
1 in 1 billion. The combination of all thirteen STRs shown in Table 15.1 typically produces frequencies of occurrence
that measure in the range of 1 in 575 trillion for Caucasian Americans and 1 in 900 trillion for African Americans.
Several commercially available kits allow forensic scientists to profile STRs in the kinds of combinations cited here.
SEX IDENTIFICATION USING STRs
Manufacturers of commercial STR kits typically used by crime laboratories provide one additional piece of useful
information along with STR types: the sex of the DNA contributor. The focus of attention here is the amelogenin gene
located on both the X and Y chromosomes. This gene, which is actually the gene for tooth pulp, has an interesting
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characteristic in that it is shorter by six bases in the X chromosome than in the Y chromosome. Hence, when the
amelogenin gene is amplified by PCR and separated by electrophoresis, males, who have an X and a Y chromosome,
show two bands; females, who have two X chromosomes, have just one band. Typically, these results are obtained in
conjunction with STR types.
FIGURE 15-19 A triplex system containing three loci: FGA, vWA, and
D3S1358, indicating a match between the questioned and the
standard/reference stains.
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Another tool in the arsenal of the DNA analyst is the ability to type STRs located on the Y chromosome. The Y
chromosome is male specific and is always paired with an X chromosome. More than twenty Y-STR markers have
been identified, and a commercial kit allows for the characterization of seventeen Y chromosome STRs. When is it
advantageous to seek out Y-STR types? Generally, Y-STRs are useful for analyzing blood, saliva, or a vaginal swab
that is a mix originating from more than one male. For example, Y-STRs prove useful when multiple males are
involved in a sexual assault.
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Y-STRs
Short tandem repeats located on the human Y chromosome.
Keep in mind that STR types derived from the Y chromosome originate only from this single male chromosome. A
female subject, with her XX chromosome pattern, does not contribute any DNA information. Also, unlike a
conventional STR analysis that is derived from two chromosomes and typically shows two bands or peaks, a Y-STR
has only one band or peak for each STR type.
WebExtra 15.11
See the Electropherogram Record from One Individual’s DNA www.mycrimekit.com
WebExtra 15.12
An Animation Depicting Y-STRs www.mycrimekit.com
FIGURE 15-20 Appropriate primers flanking the repeat units of a DNA
segment must be selected and put into place to initiate the PCR process.
FIGURE 15-21 STR profile for 15 loci.
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H. Edward Grotjan, Ph.D.
For example, the traditional STR DNA pattern may be overly complex when a vaginal swab contains the semen of two
males. Each STR type would be expected to show four bands, two from each male. Also complicating the appearance
of the DNA profile may be the presence of DNA from skin cells from the walls of the vagina. In this circumstance,
homing in on the Y chromosome greatly simplifies the appearance and interpretation of the DNA profile. Thus, when
presented with a DNA mixture of two males and one female, each STR type would be expected to show six bands.
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However, the same mixture subjected to Y-STR analysis would show only two bands (one band for each male) for
each Y-STR type.
TABLE 15.1 Thirteen CODIS STRs and Their Probability of Identities
STR AFRICAN AMERICAN U.S. CAUCASIAN
D3S1358 0.094 0.075
vWA 0.063 0.062
FGA 0.033 0.036
TH01 0.109 0.081
TPOX 0.090 0.195
CSF1PO 0.081 0.112
D5S818 0.112 0.158
D13S317 0.136 0.085
D7S820 0.080 0.065
D8S1179 0.082 0.067
D21S11 0.034 0.039
D18S51 0.029 0.028
D16S539 0.070 0.089
Source: The Future of Forensic DNA Testing: Predictions of the Research and Development Working Group . (Washington, DC: National Institute
of Justice, Department of Justice, 2000), p. 41.
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SIGNIFICANCE OF DNA TYPING
STR DNA typing has become an essential and basic investigative tool in the law enforcement community. The
technology has progressed at a rapid rate and in only a few years has surmounted numerous legal challenges so that
DNA typing is now vital evidence for resolving violent crimes and sex offenses. DNA evidence is impartial,
implicating the guilty and exonerating the innocent.
In a number of well-publicized cases, DNA evidence has exonerated individuals who have been wrongly convicted
and imprisoned. The importance of DNA analyses in criminal investigations has also placed added burdens on crime
laboratories to improve their quality-assurance procedures and to ensure the correctness of their results. In fact, in
several well-publicized instances, the accuracy of DNA tests conducted by government-funded laboratories has been
called into question.
CLOSER ANALYSIS CAPILLARY ELECTROPHORESIS
Capillary electrophoresis has emerged as the preferred technology for characterization of STRs. Capillary
electrophoresis is carried out in a thin glass column. As illustrated in the figure, each end of the column is immersed in
a reservoir of buffer liquid that also holds electrodes (coated with platinum) to supply high-voltage energy. The column
is coated with a gel polymer, and the DNA-containing sample solution is injected into one end of the column with a
syringe. The STR fragments then move through the column under the influence of an electrical potential at a speed that
is related to the length of the STR fragments. The other end of the column is connected to a detector that tracks the
separated STRs as they emerge from the column. As the DNA peaks pass through the detector, they are recorded on a
display known as an electropherogram .
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The separation of DNA segments is carried out on the interior wall of a glass capillary tube coated with a gel polymer
and kept at a constant voltage. The size of the DNA fragments determines the speed at which they move through the
column. This figure illustrates the separation of three sets of STRs (called triplexing ).
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Quick Review
• Short tandem repeats (STRs) are locations on the chromosome that contain short sequences that repeat
themselves within the DNA molecule. They serve as useful markers for identification because they are found in
great abundance throughout the human genome.
• The entire strand of an STR is very short: less than 450 bases long. This makes STRs much less susceptible to
degradation, and they are often recovered from bodies or stains that have been subjected to extreme
decomposition.
• The more STRs one can characterize, the smaller the percentage of the population from which a particular
combination of STRs can emanate. This gives rise to the concept of multiplexing, in which the forensic scientist
can simultaneously extract and amplify a combination of STRs.
• With STRs, as few as eighteen DNA-containing cells are required for analysis.
Mitochondrial DNA
Typically, when one describes DNA in the context of a criminal investigation, the DNA is assumed to be the DNA in
the nucleus of a cell. Actually, a human cell contains two types of DNA: nuclear and mitochondrial. The first
constitutes the twenty-three pairs of chromosomes in the nuclei of our cells. Each parent contributes to the genetic
makeup of these chromosomes. Mitochondrial DNA (mtDNA), on the other hand, is found outside the nucleus of the
cell and is inherited solely from the mother.
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Mitochondria are cell structures found in all human cells. They are the power plants of the body, providing about 90
percent of the energy that the body needs to function. A single mitochondrion contains several loops of DNA, all of
which are involved in energy generation. Further, because each cell in our bodies contains hundreds to thousands of
mitochondria, there are hundreds to thousands of mtDNA copies in a human cell. This compares to just one set of
nuclear DNA located in that same cell.
mitochondria
Small structures outside the nucleus that supply energy to a cell.
Forensic scientists rely on mtDNA to identify a subject when nuclear DNA is significantly degraded, as in the case of
charred remains, or when nuclear DNA may be present in only very small quantities (such as in a hair shaft).
Interestingly, when authorities cannot obtain a reference sample from an individual who may be long deceased or
missing, an mtDNA reference sample can be obtained from any maternally related relative. However, this also means
that all individuals of the same maternal lineage will be indistinguishable by mtDNA analysis.
Although mtDNA analysis is significantly more sensitive than nuclear DNA profiling, forensic analysis of mtDNA is
more rigorous, time consuming, and costly than nuclear DNA profiling. For this reason, only a handful of public and
private forensic laboratories receive evidence for mtDNA determination. The FBI Laboratory strictly limits the types
of cases in which it will apply mtDNA technology.
WebExtra 15.13
See How We Inherit Our Mitochondrial DNA www.mycrimekit.com
WebExtra 15.14
Look into the Structure of Mitochondrial DNA and See How It’s Used for DNA Typing www.mycrimekit.com
One of the most publicized cases performed on human remains was the identification of the individual buried in the
tomb of the Vietnam War’s unknown soldier. The remains lying in the tomb were believed to belong to 1st Lt. Michael
J. Blassie, whose A-37 warplane was shot down near An Loc, South Vietnam, in 1972. In 1984, the US Army Central
Identification Laboratory failed to identify the remains by physical characteristics, personal artifacts, or blood-typing
results. The remains were subsequently placed in the tomb. In 1998, at the insistence of the Blassie family, the remains
were disinterred for mtDNA analysis and the results were compared to references from seven families thought to be
associated with the case. The remains in the tomb were subsequently analyzed and confirmed to be consistent with
DNA from Lt. Blassie’s family.
CLOSER ANALYSIS FORENSIC ASPECTS OF MITOCHONDRIAL
DNA
As discussed previously, nuclear DNA is composed of a continuous linear strand of nucleotides (A, C, G , and T). By
contrast, mtDNA is constructed in a circular or loop configuration. Each loop contains enough A, C, G , and T
(approximately 16,569 total nucleotides) to make up thirty-seven genes involved in mitochondrial energy generation.
Two regions of mtDNA have been found to be highly variable in the human population. These two regions have been
designated hypervariable region I (HV1) and hypervariable region II (HV2), as shown in the figure. Again, the process
for analyzing HV1 and HV2 is tedious. It involves generating many copies of these DNA hypervariable regions by
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PCR and then determining the order of the A-T-C-G bases constituting the hypervariable regions. This process is
known as sequencing . The FBI Laboratory, the Armed Forces DNA Identification Laboratory, and other laboratories
have collaborated to compile an mtDNA population database containing the base sequences from HV1 and HV2.
Once the sequences of the hypervariable regions from a case sample are obtained, most laboratories simply report the
number of times these sequences appear in the mtDNA database maintained by the FBI. The mtDNA database
contains about five thousand sequences. This approach permits an assessment of how common or rare an observed
mtDNA sequence is in the database.
Interestingly, many of the sequences that have been determined in case work are unique to the existing database, and
many types are present at frequencies of no greater than 1 percent in the database. Thus, it is often possible to
demonstrate how uncommon a particular mtDNA sequence is. However, even under the best circumstances, mtDNA
typing does not approach STR analysis in its discrimination power. Thus, mtDNA analysis is best reserved for samples
for which nuclear DNA typing is simply not possible.
The first time mtDNA was admitted as evidence in a US court was in 1996 in the case of State of Tennessee v. Paul
Ware . Here, mtDNA was used to link two hairs recovered from the crime scene to the defendant. Interestingly, in this
case, blood and semen evidence were absent. Mitochondrial DNA analysis also plays a key role in the identification of
human remains. An abundant amount of mtDNA is generally found in skeletal remains. Importantly, mtDNA reference
samples are available from family members sharing the same mother, grandmother, great-grandmother, and so on.
Every cell in the body contains hundreds of mitochondria, which provide energy to the cell. Each mitochondrion
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contains numerous copies of DNA shaped in the form of a loop. Distinctive differences between individuals in their
mitochondrial DNA makeup are found in two specific segments of the control region on the DNA loop, known as HV1
and HV2.
CASE FILES
In the fall of 1979, a 61-year-old patient wandered away from a US Department of Veterans Affairs medical facility.
Despite an extensive search, authorities never located the missing man. More than ten years later, a dog discovered a
human skull in a wooded area near the facility. DNA Analysis Unit II of the FBI Laboratory received the case in the
winter of 1999. The laboratory determined that the mitochondrial DNA profile from the missing patient’s brother
matched the mitochondrial DNA profile from the recovered skull and provided the information to the local medical
examiner. Subsequently, the remains were declared to be those of the missing patient and returned to the family for
burial.
Source: FBI Law Enforcement Bulletin 78 (2002): 21.
Quick Review
• Mitochondrial DNA is located outside the cell’s nucleus and is inherited from the mother.
• Mitochondria are cell structures found in all human cells. They provide most of the energy that the body needs
to function.
• Mitochondrial DNA typing does not approach STR analysis in its discrimination power and thus is best
reserved for analyzing samples, such as hair, for which STR analysis is not possible.
Combined DNA Index System (CODIS)
Perhaps the most significant investigative tool to arise from a DNA-typing program is CODIS (Combined DNA Index
System), a computer software program developed by the FBI that maintains local, state, and national databases of
DNA profiles from convicted offenders, unsolved crime-scene evidence, and profiles of missing people. CODIS
allows crime laboratories to compare DNA types recovered from crime-scene evidence to those of convicted sex
offenders and other convicted criminals.
Thousands of CODIS matches have linked serial crimes to each other and have solved crimes by allowing
investigators to match crime-scene evidence to known convicted offenders. This capability is of tremendous value to
investigators in cases in which the police have not been able to identify a suspect. The CODIS concept has already had
a significant impact on police investigations in various states, as shown in the Case Files feature on page 401 .
Quick Review
• CODIS is a computer software program developed by the FBI that maintains local, state, and national
databases of DNA profiles from convicted offenders, unsolved crime-scene evidence, and profiles of missing
people.
Collection and Preservation of Biological Evidence for DNA Analysis
Since the early 1990s, the advent of DNA profiling has vaulted biological crime-scene evidence to a stature of
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importance that is eclipsed only by the fingerprint. In fact, the high sensitivity of DNA determinations has even
changed the way police investigators define biological evidence.
Just how sensitive is STR profiling? Forensic analysts using currently accepted protocols can reach sensitivity levels as
low as 125 picograms . Interestingly, a human cell has an estimated 7 picograms of DNA, which means that only
eighteen DNA-bearing cells are needed to obtain an STR profile. With this technology in hand, the horizon of the
criminal investigator extends beyond the traditional dried blood or semen stain to include stamps and envelopes licked
with saliva, a cup or can that has touched a person’s lips, chewing gum, the sweat band of a hat, or a bedsheet
containing dead skin cells. Likewise, skin cells, or epithelial cells , transferred onto the surface of a weapon, the
interior of a glove, a pen, or any object recovered from a crime scene have yielded DNA results.
5
The phenomenon of
transferring DNA via skin cells onto the surface of an object is called touch DNA . Again, keep in mind that, in theory,
only 18 skin cells deposited on an object are required to obtain a DNA profile.
epithelial cells
The outer layer of skin cells.
touch DNA
DNA from skin cells transferred onto the surface of an object by simple contact.
Modifications to the STR technology can readily extend the level of detection down to nine or even fewer cells. A
quantity of DNA that is below the normal level of detection is defined as a low copy number . However, analysts must
take extraordinary care in analyzing low copy number DNA and often may find that courts will not allow this data to
be admissible in a criminal trial.
low copy number
Fewer than 18 DNA-bearing cells.
COLLECTION OF BIOLOGICAL EVIDENCE
Before an investigator becomes enamored of the wonders of DNA, he or she should first realize that the crime scene
must still be treated in the traditional manner. Before the collection of evidence begins, biological evidence should be
photographed close up, and its location relative to the entire crime scene must be recorded through notes, sketches, and
photographs. If the shape and position of bloodstains may provide information about the circumstances of the crime,
an expert must immediately evaluate the blood evidence. The significance of the position and shape of bloodstains can
best be ascertained when the expert has an on-site overview of the entire crime scene and can better reconstruct the
movement of the individuals involved. The blood pattern should not be disturbed to collect DNA evidence before this
phase of the investigation is completed.
The evidence collector must handle all body fluids and biologically stained materials with a minimum of personal
contact. All body fluids must be assumed to be infectious; hence, wearing disposable latex gloves while handling the
evidence is required. Latex gloves also significantly reduce the possibility that the evidence collector will contaminate
the evidence. These gloves should be changed frequently during the evidence-collection phase of the investigation.
Safety considerations and avoidance of contamination also call for the wearing of face masks, shoe covers, and
possibly coveralls.
Blood has great evidential value when a transfer between a victim and suspect can be demonstrated. For this reason, all
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clothing from both victim and suspect should be collected and sent to the laboratory for examination. This procedure
must be followed even when the presence of blood on a garment is not obvious to the investigator. Laboratory search
procedures are far more revealing and sensitive than any that can be conducted at the crime scene. In addition, blood
should also be searched for in less-than-obvious places. For example, the criminal may have wiped his or her hands on
materials not readily apparent to the investigator. Investigators should look for towels, handkerchiefs, or rags that may
have been used and then hidden, and should also examine floor cracks or other crevices that may have trapped blood.
CASE FILES
In 1990, a series of attacks on elderly victims was committed in Golds-boro, North Carolina, by an unknown
individual dubbed the Night Stalker. During one such attack in March, an elderly woman was brutally sexually
assaulted and almost murdered. Her daughter’s early arrival home saved the woman’s life. The suspect fled, leaving
behind materials intended to burn the residence and the victim in an attempt to conceal the crime.
In July 1990, another elderly woman was sexually assaulted and murdered in her home. Three months later, a third
elderly woman was sexually assaulted and stabbed to death. Her husband was also murdered. Although their house
was set alight in an attempt to cover up the crime, fire and rescue personnel pulled the bodies from the house before it
was engulfed in flames. DNA analysis of biological evidence collected from vaginal swabs from the three sexual
assault victims enabled authorities to conclude that the same perpetrator had committed all three crimes. However,
there was no suspect.
More than ten years after these crimes were committed, law enforcement authorities retested the biological evidence
from all three cases using newer DNA technology and entered the DNA profiles into North Carolina’s DNA database.
The DNA profile developed from the crime-scene evidence was compared to thousands of convicted-offender profiles
already in the database.
In April 2001, a “cold hit” was made: The DNA profiles was matched to that of an individual in the convicted-offender
DNA database. The perpetrator had been convicted of shooting into an occupied dwelling, an offense that requires
inclusion of the convict’s DNA in the North Carolina DNA database. The suspect was brought into custody for
questioning and was served with a search warrant to obtain a sample of his blood. That sample was analyzed and
compared to the crime-scene evidence, confirming the DNA database match. When confronted with the DNA
evidence, the suspect confessed to all three crimes.
Source: National Institute of Justice, “Using DNA to Solve Cold Cases” (NIJ Special Report), July 2002, https://www.ncjrs.gov/pdffiles1
/nij/194197.pdf
PACKAGING OF BIOLOGICAL EVIDENCE
Biological evidence should not be packaged in plastic or airtight containers because accumulation of residual moisture
could contribute to the growth of DNA-destroying bacteria and fungi. Each stained article should be packaged
separately in a paper bag or a well-ventilated box. A red bio-hazard label must be attached to each container. If
feasible, the entire stained article should be packaged and submitted for examination. If this is not possible, dried blood
is best removed from a surface with a sterile cotton-tipped swab lightly moistened with distilled water from a dropper
bottle.
A portion of the unstained surface material near the recovered stain must likewise be removed or swabbed and placed
in a separate package. This is known as a substrate control . The forensic examiner might use the substrate swab to
confirm that the results of the tests performed were brought about by the stain and not by the material on which it was
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deposited. However, this practice is normally not necessary when DNA determinations are carried out in the
laboratory. It is critical that the collection swabs must not be packaged in a wet state. After collection, a swab must be
air-dried for approximately five to ten minutes. Then it is best to place it in a swab box (see Figure 15-22 ), which has a
circular hole to allow air circulation. The swab box can then be placed in a paper or manila envelope.
substrate control
An unstained object adjacent to an area on which biological material has been deposited.
FIGURE 15-22 Air-dried swabs are placed in a swab box for delivery to
the forensic laboratory.
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Courtesy Tri-Tech, Inc., Southport, NC, www.tritechusa.com
TABLE 15.2 Location and Sources of DNA at Crime Scenes
EVIDENCE
POSSIBLE LOCATION OF DNA ON THE
EVIDENCE
SOURCE OF DNA
Baseball bat or similar
weapon
Handle, end Sweat, skin, blood, tissue
Hat, bandanna, or mask Inside Sweat, hair, dandruff
Eyeglasses Nose or ear pieces, lens Sweat, skin
Facial tissue, cotton swab Surface area
Mucus, blood, sweat, semen, ear
wax
Dirty laundry Surface area Blood, sweat, semen
Toothpick Tips Saliva
Used cigarette Cigarette butt Saliva
Stamp or envelope Licked area Saliva
Tape or ligature Inside/outside surface Skin, sweat
Bottle, can, or glass Sides, mouthpiece Saliva, sweat
Used condom Inside/outside surface Semen, vaginal and/or rectal cells
Blanket, pillow, sheet Surface area Sweat, hair, semen, urine, saliva
“Through and through”
bullet
Outside surface Blood, tissue
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EVIDENCE
POSSIBLE LOCATION OF DNA ON THE
EVIDENCE
SOURCE OF DNA
Bite mark Person’s skin or clothing Saliva
Fingernail, partial fingernail Scrapings Blood, sweat, tissue
Source: National Institute of Justice, US Department of Justice.
All packages containing biological evidence should be refrigerated or stored in a cool location out of direct sunlight
until delivery to the laboratory. However, one common exception is blood mixed with soil. Microbes present in soil
rapidly degrade DNA. Therefore, blood in soil must be stored in a clean glass or plastic container and immediately
frozen.
OBTAINING DNA REFERENCE SPECIMENS
Biological evidence attains its full forensic value only when an analyst can compare each of its DNA types to known
DNA samples collected from victims and suspects. For this purpose, at least 7 cc of whole blood should be drawn from
individuals by a qualified medical professional. The blood sample should be collected in a sterile vacuum tube
containing the preservative EDTA (ethylenediamine tetraacetic acid). In addition to serving as a preservative, EDTA
inhibits the activity of enzymes that degrade DNA. The tubes must be kept refrigerated (not frozen) while awaiting
transportation to the laboratory. In addition to extracting blood, there are other ways of obtaining standard/reference
DNA specimens. The least intrusive method for obtaining a DNA standard/reference, one that nonmedical personnel
can readily use, is the buccal swab . Cotton swabs are inserted into the subject’s mouth, and the inside of the cheek is
vigorously swabbed, resulting in the transfer of buccal cells onto the swab.
buccal cells
Cells from the inner cheek lining.
With the increasing need for collection and analysis of DNA samples in forensic investigations, collection and
long-term storage of DNA has become an important consideration. FTA brand paper is a type of commercially
available filter paper loaded with a mix of reagents on which DNA samples can be stored. An FTA paper card has been
impregnated with a chemical that protects DNA from bacterial enzyme breakdown. The fibers of the paper can entrap
the DNA for at least ten years without refrigeration, allowing it to be easily stored. Figure 15-23 illustrates the
collection of a buccal swab and its transfer onto an FTA card for storage.
If an individual is not available to give a DNA standard/reference sample, some interesting alternative sources are
available, including the individual’s toothbrush, comb or hairbrush, razor, soiled laundry, used cigarette butts, and
earplugs. Any of these items may contain a sufficient quantity of DNA for typing. Interestingly, as investigators
worked to identify the remains of victims of the World Trade Center attack on September 11, 2001, the families of the
missing were asked to supply the New York City DNA Laboratory with these types of items in an effort to match
recovered DNA with human remains.
CONTAMINATION OF DNA EVIDENCE
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One key concern while collecting a DNA-containing specimen is contamination. Contamination can occur by
introducing foreign DNA onto a stain through coughing or sneezing during the collection process, or there can be a
transfer of DNA when items of evidence are incorrectly placed in contact with each other during packaging.
Fortunately, an examination of DNA band patterns in the laboratory readily reveals the presence of contamination. For
example, with an STR, one will expect to see a two-band pattern. More than two bands suggests a mixture of DNA
from more than one source.
Crime-scene investigators can take some relatively simple steps to minimize the contamination of biological evidence:
1. Change gloves before handling each new piece of evidence.
2. Collect a substrate control for possible subsequent laboratory examination.
3. Pick up small items of evidence such as cigarette butts and stamps with clean forceps. Use disposable forceps
so that they can be discarded after a single evidence collection.
4. Always package each item of evidence in its own well-ventilated container.
A common occurrence at crime scenes is to suspect the presence of blood but not be able to observe any with the
naked eye. In these situations, the common test of choice is luminol or Bluestar. Interestingly, luminol and Bluestar do
not inhibit the ability to detect and characterize STRs.
2
Therefore, they can be used to locate traces of blood and areas
that have been washed nearly free of blood without compromising the potential for DNA typing.
Quick Review
• Packaging of bloodstained evidence in plastic or airtight containers must be avoided because the accumulation
of residual moisture could contribute to the growth of DNA-destroying bacteria and fungi. Each stained article
should be packaged separately in a paper bag or in a well-ventilated box.
• The least intrusive method for obtaining a DNA standard/reference is the buccal swab. In this procedure,
cotton swabs are inserted into the subject’s mouth, and the inside of the cheek is vigorously swabbed, resulting
in the transfer of cells from the inner cheek lining onto the swab.
WebExtra 15.15
DNA Forensics www.mycrimekit.com
WebExtra 15.16
Step into the Role of the First Responding Officer at a Sexual Assault Scene www.mycrimekit.com
WebExtra 15.17
Assume the Duties of an Evidence Collection Technician at a Sexual Assault Scene www.mycrimekit.com
Virtual Lab DNA Analysis
To perform a virtual DNA analysis go to www.pearsoncustom.com/us/vlm/
Virtual Lab DNA Analysis
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To perform a virtual bloodstain analysis, go to www.pearsoncustom.com/us/vlm/
FIGURE 15-23 (a) A buccal swab is collected by rubbing each cheek for
15 seconds. (b) A protective film is peeled off the FTA card. (c) The swab is
snapped in place against the FTA paper. (d) The FTA card is removed
from the collection device and stored.
Courtesy GE Healthcare Bio-Sciences Corp. (GEHC), Piscataway, NJ, www.whatman.com
CASE FILES
A woman alleged that she had been held in an apartment against her will and sexually assaulted by a male friend.
During the course of the assault, a contact lens was knocked from the victim’s eye. After the assault, she escaped, but
out of fear from threats made by her attacker, she did not report the assault to the police for three days. When the
police examined the apartment, they noted that it had been thoroughly cleaned. A vacuum cleaner bag was seized for
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examination, and several pieces of material resembling fragments of a contact lens were discovered within the bag.
In the laboratory, approximately 20 nanograms of human DNA were recovered from the contact lens fragments.
Because cells from both a person’s eyeballs and the interior of the eyelids are naturally replaced every 6 to 24 hours,
both were potential sources for the DNA found. The DNA profile originating from the fragments matched the victim,
thus corroborating the victim’s account of the crime. The estimated frequency of occurrence in the population for the
nine matching STRs is approximately 1 in 850 million. The suspect subsequently pleaded guilty to the offense.
*
STR Locus Victim’s DNA Type Contact Lens
D3S1358 15,18 15,18
FGA 24,25 24,25
vWA 17,17 17,17
THO1 6,7 6,7
F13A1 5,6 5,6
fes/fps 11,12 11,12
D5S818 11,12 11,12
D13S317 11,12 11,12
D7S820 10,12 10,12
*
Based on information contained in R. A. Wickenheiser and R. M. Jobin, “Comparison of DNA Recovered from a Contact Lens Using PCR DNA
Typing.” Canadian Society of Forensic Science Journal 32 (1999): 67.
CHAPTER REVIEW
• An antibody reacts or agglutinates only with its specific antigen. The concept of specific antigen-antibody
reactions has been applied to techniques for the detection of commonly abused drugs in blood and urine.
• Every red blood cell contains either an A antigen, a B antigen, both antigens, or no antigen (this is called type
O). The type of antigen on one’s red blood cells determines one’s A-B-O blood type. Persons with type A blood
have A antigens on their red blood cells, those with type B blood have B antigens, those with type AB blood
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have both antigens, and those with type O blood have no antigens on their red blood cells.
• To produce antibodies capable of reacting with drugs, a specific drug is combined with a protein, and this
combination is injected into an animal such as a rabbit. This drug-protein complex acts as an antigen, stimulating
the animal to produce antibodies. The recovered blood serum of the animal will now contain antibodies that are
specific or nearly specific to the drug.
• The criminalist must be prepared to answer the following questions when examining dried blood: (1) Is it
blood? (2) From what species did the blood originate? (3) If the blood is of human origin, how closely can it be
associated to a particular individual?
• The determination that a substance is blood is best made by means of a preliminary color test. A positive result
from the Kastle-Meyer color test is highly indicative of blood.
• The luminol and Bluestar tests are used to search out trace amounts of blood located at crime scenes.
• The precipitin test uses antisera, normally derived from rabbits that have been injected with the blood of a
known animal, to determine the species origin of a questioned bloodstain.
• The best way to locate and characterize a seminal stain is to perform the acid phosphatase color test.
• The presence of spermatozoa is a unique identifier of semen. Also, the protein called prostate-specific antigen
(PSA), also known as p30 , is useful in combination with the acid phosphatase color test for characterizing a
sample stain as semen.
• Forensic scientists can link seminal material to an individual by DNA typing.
• A sexual assault victim should undergo a medical examination as soon as possible after the assault. At that
time clothing, hairs, and vaginal and rectal swabs can be collected for subsequent laboratory examination.
• The persistence of seminal constituents in the vagina may help determine the time of an alleged sexual attack.
• The gene is the basic unit of heredity. A chromosome is a threadlike structure in the cell nucleus along which
the genes are located.
• Most human cells contain forty-six chromosomes, arranged in twenty-three mated pairs. The only exceptions
are the human reproductive cells, the egg and sperm, which contain twenty-three unmated chromosomes each.
• During fertilization, a sperm and an egg combine so that each contributes twenty-three chromosomes to form
the new cell, or zygote , that develops into the offspring.
• An allele is any of several alternative forms of genes that influence a given characteristic and that are aligned
with one another on a chromosome pair.
• A heterozygous gene pair is made up of two different alleles; a homozygous gene pair is made up of two
similar alleles.
• When two different genes are inherited, the characteristic in the dominant gene’s code will be expressed. The
characteristic in the recessive gene’s code will remain hidden.
• The gene is the fundamental unit of heredity. Each gene is composed of DNA specifically designed to control
the genetic traits of our cells.
• DNA is constructed as a very large molecule made of a linked series of repeating units called nucleotides .
• Four types of bases are associated with the DNA structure: adenine (A) , guanine (G) , cytosine (C) , and thymine
(T) .
• The bases on each strand of DNA are aligned in a double-helix configuration so that adenine pairs with
thymine and guanine pairs with cytosine. This concept is known as complementary base pairing .
• The order in which the base pairs are arranged defines the role and function of a DNA molecule.
• DNA replication begins with the unwinding of the DNA strands in the double helix. The double helix is
re-created as the nucleotides are assembled in the proper order ( A with T and G with C ). Two identical copies of
DNA emerge from the process.
• PCR (polymerase chain reaction) is a technique for replicating or copying a portion of a DNA strand outside a
living cell.
• Short tandem repeats (STRs) are locations on the chromosome that contain short sequences that repeat
themselves within the DNA molecule. They serve as useful markers for identification because they are found in
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great abundance throughout the human genome.
• The entire strand of an STR is very short: less than 450 bases long. This makes STRs much less susceptible to
degradation, and they are often recovered from bodies or stains that have been subjected to extreme
decomposition.
• The more STRs one can characterize, the smaller the percentage of the population from which a particular
combination of STRs can emanate. This gives rise to the concept of multiplexing, in which the forensic scientist
can simultaneously extract and amplify a combination of STRs.
• With STRs, as few as eighteen DNA-containing cells are required for analysis.
• Mitochondrial DNA is located outside the cell’s nucleus and is inherited from the mother.
• Mitochondria are cell structures found in all human cells. They provide most of the energy that the body needs
to function.
• Mitochondrial DNA typing does not approach STR analysis in its discrimination power and thus is best
reserved for analyzing samples, such as hair, for which STR analysis is not possible.
• CODIS is a computer software program developed by the FBI that maintains local, state, and national
databases of DNA profiles from convicted offenders, unsolved crime-scene evidence, and profiles of missing
people.
• Packaging of bloodstained evidence in plastic or airtight containers must be avoided because the accumulation
of residual moisture could contribute to the growth of DNA-destroying bacteria and fungi. Each stained article
should be packaged separately in a paper bag or in a well-ventilated box.
• The least intrusive method for obtaining a DNA standard/ reference is the buccal swab. In this procedure,
cotton swabs are inserted into the subject’s mouth and the inside of the cheek is vigorously swabbed, resulting in
the transfer of cells from the inner cheek lining onto the swab.
KEY TERMS
acid phosphatase, 373
agglutination, 372
allele, 373
antibody, 372
antigen, 373
antiserum, 372
aspermia, 379
buccal cells, 402
chromosome, 385
deoxyribonucleic acid (DNA), 371
egg, 385
epithelial cells, 400
gene, 385
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heterozygous, 386
homozygous, 386
locus, 386
low copy number, 400
mitochondria, 397
multiplexing, 392
nucleotide, 387
oligospermia, 379
plasma, 371
polymerase chain reaction (PCR), 389
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