Case Study: Use the Internet and Chapter 11 of your text to research the murder of JonBenet Ramsey. Use 3 references1. Case Summary – In a narrative format, discuss the key facts and critical issues presented in the case. Minimum word count is 500 words.2. Case Analysis – Based on your research, determine who you believe the killer to be and why. Give at least five reasons/factors (physical evidence) to support your conclusion.3. Executive Decisions – As lead investigator in the case, you would need to hold a debriefing with your detectives. List what they did correct and any mistakes that were made. What should be done to ensure the mistakes are not repeated in the future?headline news
The Green River Killer
This case takes its name from the Green
G flows through Washington state and empties
River, which
into PugetO
Sound in Seattle. In 1982, within six months the
bodies ofR
five females were discovered in or near the river.
Most of the
D victims were known prostitutes who were
strangled
O and apparently raped. As police focused their
attention
N on an area known as Sea-Tac Strip, a haven for
prostitutes, girls mysteriously disappeared with
,
increasing frequency. By the end of 1986, the body
count in the Seattle region rose to 40, all of whom
J
were believed to have been murdered by the Green
E
River Killer. As the investigation pressed on into
S the police renewed their interest in one
1987,
S
suspect,
Gary Ridgway, a local truck painter.
IRidgway had been known to frequent the Sea-Tac
CStrip. Interestingly, in 1984 Ridgway had actually
A passed a lie detector test. Now with a search
warrant in hand, police searched the Ridgway
L residence and also obtained hair and saliva
E samples from Ridgway. Again, insufficient
evidence caused Ridgway to be released
I
from custody. With the exception of one killing in
G
1998, the murder spree stopped in 1990, and the case remained
H
dormant for nearly ten years. But the advent of DNA testing brought renewed
vigor to the investigation. In 2001, semen samples collected from three early victims of the
1 had been collected in 1987. The DNA profiles
Green River Killer were compared to Ridgway’s saliva that
8 link to Ridgway was made by the location of
matched and the police had their man. An added forensic
minute amounts of spray paint on the clothing of six victims
7 that compared to paints collected from
Ridgway’s workplace. Ridgway avoided the death penalty
1 by confessing to the murders of 48 women.
B
U
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#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
>>>>>>>>>>>>
chapter
Learning Objectives
forensic
serology
G
O
R
D
O
N
,
J
E
S
After studying this chapter you should
be able to:
S in the blood for
• List the A-B-O antigens and antibodies found
I O
each of the four blood types: A, B, AB, and
C is typed
• Understand and describe how whole blood
A
• List and describe forensic tests used to characterize
a stain
as blood
L interactions
• Understand the concept of antigen–antibody
and how it is applied to species identification
E and drug
identification
•
I
Explain the differences between monoclonal
G and polyclonal
antibodies
H
• Contrast chromosomes and genes
• Learn how the Punnett square is used to determine
the
1
genotypes and phenotypes of offspring
8
• List the laboratory tests necessary to characterize
seminal stains
7
• Explain how suspect blood and semen stains
1 are to be
properly preserved for laboratory examination
ISBN 1-256-36593-9
•
B
Describe the proper collection of physicalUevidence in a rape
investigation
10
KEY TERMS
acid phosphatase
agglutination
allele
antibody
antigen
antiserum
aspermia
chromosome
deoxyribonucleic
acid (DNA)
egg
erythrocyte
gene
genotype
hemoglobin
heterozygous
homozygous
hybridoma cells
locus
luminol
monoclonal antibodies
oligospermia
phenotype
plasma
polyclonal antibodies
precipitin
serology
serum
sperm
X chromosome
Y chromosome
zygote
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242
CHAPTER 10
DNA
Abbreviation for deoxyribonucleic
acid—the molecules carrying the
body’s genetic information; DNA is
double stranded in the shape of a
double helix
In 1901, Karl Landsteiner announced one of the most significant discoveries of the 20th century—
the typing of blood—a finding that 29 years later earned him a Nobel Prize. 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 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 had the key for properly matching the blood
of a donor to a recipient. 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
the millions of lives it has since saved. Meanwhile, Landsteiner’s findings had opened up a completely new field of research in the biological sciences. Others began to pursue the identification of
additional characteristics that couldGfurther differentiate blood. By 1937, the Rh factor in blood was
demonstrated, and shortly thereafter,
Onumerous blood factors or groups were discovered. More than
a hundred different blood factors have been shown to exist. However, the ones in the A-B-O system
R matching a donor and recipient for a transfusion.
are still the most important for properly
Until the early 1990s, forensic
Dscientists 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 to the
O
forensic scientist was that in theory no two individuals, except for identical twins, could be expected to have the same combination
N 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 high frequency of occurrence of bloodstains at
crime scenes, especially crimes of the most serious nature—that is, homicides, assaults, and
rapes. Consider, for example, a transfer
J of blood between the victim and assailant during a struggle; that is, the victim’s blood is transferred to the suspect’s garment, or vice versa. If the criminalist could individualize human E
blood by identifying all of its known factors, the result would
be evidence of the strongest kind S
for linking the suspect to the crime scene.
The advent of DNA technology has dramatically altered the approach of forensic scientists toS and other biological evidence. The search for genetically
ward individualization of bloodstains
controlled blood factors in bloodstains
I has been abandoned in favor of characterizing biological
evidence by select regions of our deoxyribonucleic acid (DNA). The individualization of dried
C now a reality, has significantly altered the role that crime labblood and other biological evidence,
oratories play in criminal investigations.
A As we will learn in the next chapter, the high sensitivity
of DNA analysis has even altered the type of materials collected from crime scenes in the search
for DNA. The next chapter is devoted to discussing recent breakthroughs in associating blood and
semen stains with a single individual
L through characterization of DNA. This chapter focuses on
underlying biological concepts that forensic scientists historically relied on as they sought to charE
acterize and individualize biological evidence before the dawning of the age of DNA.
The Nature of
plasma
The fluid portion of unclotted
blood
erythrocyte
A red blood cell
serum
The liquid that separates from the
blood when a clot is formed
I
G
Blood
H
Antigens and Antibodies
Functionally, red blood cells transport oxygen from the lungs to the body tissues and in turn 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
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
ISBN 1-256-36593-9
The word blood actually refers to a highly complex mixture of cells, enzymes, proteins, and inorganic substances. The fluid portion of blood is called plasma. Plasma is composed principally
1 of blood content. Suspended in the plasma are solid materiof water and accounts for 55 percent
als consisting chiefly of cells—that
8 is, red blood cells (erythrocytes), white blood cells (leukocytes), and platelets. The solid portion of blood accounts for 45 percent of its content. Blood clots
7 as fibrin traps and enmeshes the red blood cells. If one were
when a protein in the plasma known
to remove the clotted material, a pale
1 yellowish liquid known as serum would be left.
Obviously, considering the complexity of blood, any discussion of its function and chemistry
B beyond the scope of this text. It is certainly far more relewould have to be extensive, extending
vant at this point to concentrate our
U discussion on the blood components that are directly pertinent to the forensic aspects of blood identification—the red blood cells and the blood serum.
FORENSIC SEROLOGY
are millions of characteristic chemical structures called antigens. Antigens impart blood-type
characteristics to the red blood cells. Blood antigens are grouped into systems depending on their
relationship to one another. More than 15 blood antigen systems have been identified to date; of
these, the A-B-O and Rh systems are the most important.
If an individual is type A, this simply indicates 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 contain both A and B antigens. Type O individuals have neither A nor B antigens on their cells. Hence,
the presence or absence of the A and B antigens on the red blood cells determines a person’s blood
type in the A-B-O system.
Another important blood antigen has been designated as the Rh factor, or D antigen. People
with the D antigen are said to be Rh positive; those without this antigen are Rh negative. In routine
GB, and D—must be determined
blood banking, the presence or absence of the three antigens—A,
in testing for the compatibility of the donor and recipient.
O
Serum is important because it contains certain proteins known as antibodies. The fundaR exists a specific antibody.
mental principle of blood typing is that for every antigen, there
Each antibody symbol contains the prefix anti-, followed by theD
name of the antigen for which it
is specific. Hence, anti-A is specific only for A antigen, anti-B for B antigen, and anti-D for
O
D antigen. The serum-containing antibody is referred to as the antiserum, meaning a serum that
N
reacts against something (antigens).
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 immediately 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
J be attached to antigens located
on two different red blood cells. This creates a vast network of cross-linked cells usually seen as
E
clumping or agglutination (see Figure 10–1).
Let’s look a little more closely at this phenomenon. In normalSblood, shown in Figure 10–2(a),
antigens on red blood cells and antibodies coexist without destroying each other because the antiS suppose a foreign serum added
bodies present are not specific toward any of the antigens. However,
to the blood introduces a new antibody. The occurrence of a specific
I antigen–antibody reaction immediately causes the red blood cells to link together, or agglutinate, as shown in Figure 10–2(b).
C when we examine the serum
Evidently, nature has taken this situation into account because
of type A blood, we find anti-B and no anti-A. Similarly, type BA
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
A
B
AB
O
A
B
AB
Neither A nor B
antigen
A substance, usually a protein, that
stimulates the body to produce
antibodies against it
antibody
A protein that destroys or
inactivates a specific antigen;
antibodies are found in the blood
serum
antiserum
Blood serum that contains specific
antibodies
agglutination
The clumping together of red
blood cells by the action of an
antibody
L
AntibodiesEin Serum
I
Anti-B
Anti-A
G
Neither anti-A nor anti-B
Hand anti-B
Both anti-A
The reasons for the fatal consequences of mixing incompatible
1 blood during a transfusion
should now be quite obvious. For example, transfusing type A blood into a type B patient will
8 promptly with the incoming A
cause the natural anti-A in the blood of the type B patient to react
antigens, resulting in agglutination. In addition, the incoming anti-B
7 of the donor will react with
the B antigens of the patient.
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1
B
Blood Typing
The term serology is used to describe a broad scope of laboratory
U tests that use specific antigen
and serum antibody reactions. The most widespread application of serology is the typing of whole
blood for its A-B-O identity. In determining the A-B-O blood type, only two antiserums are
needed—anti-A and anti-B. For routine blood typing, both of these antiserums are commercially
available.
Table 10–1 summarizes how the identity of each of the four blood groups is established when
the blood is tested with anti-A and anti-B serum. Type A blood is agglutinated by anti-A serum;
243
serology
The study of antigen–antibody
reactions
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244
CHAPTER 10
FIGURE 10–1
A
A
A
Anti-B
G blood cells containing A antigens
Red
doOnot combine with B antibodies
R
D
O
N
,
B
B
B
B
J
E
S
B
S
Anti-B
I
C blood cells containing B antigens
Red
are
Aagglutinated or clumped together
B
in the presence of B antibodies
L
E
I
G
H
1
8
7
1
B
U
Courtesy J. C. Revy, Phototake NYC
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
ISBN 1-256-36593-9
FIGURE 10–2
(a) Microscopic view of normal red blood cells (500 ). (b) Microscopic view of agglutinated red blood cells (500 ).
FORENSIC SEROLOGY
TABLE 10–1
Identification of Blood with Known Antiserum
Anti-A Serum
Whole Blood
Anti-B Serum
Whole Blood
Antigen Present
Blood Type
A
B
A and B
Neither A nor B
A
B
AB
O
G
O
R
D
Antibody Present
O
Anti-AN
Anti-B
, anti-B
Both anti-A and
Note: shows agglutination; – shows absence of agglutination.
TABLE 10–2
Identification of Blood with Known Cells
A Cells Blood
B Cells Blood
Neither anti-A nor anti-B
Blood Type
B
A
O
AB
J
E
type B blood is agglutinated by anti-B serum; type AB blood is agglutinated by both anti-A and
anti-B; and type O blood is not agglutinated by either the anti-ASor anti-B serum.
The identification of natural antibodies present in blood offers
S another route to the determination of blood type. Testing blood for the presence of anti-A and anti-B requires using red blood
I
cells that have known antigens. Again, these cells are commercially
available. Hence, when A
cells are added to a blood specimen, agglutination occurs onlyC
in the presence of anti-A. Similarly, B cells agglutinate only in the presence of anti-B. All four A-B-O types can be identified in
A
this manner by testing blood with known A and B cells, as summarized in Table 10–2.
Note: shows agglutination; – shows absence of agglutination.
The population distribution of blood types varies with location and race throughout the
world. In the United States, a typical distribution is as follows:
O
A
B
AB
43%
42%
12%
3%
Immunoassay Techniques
L
E
I
G
H
The concept of a specific antigen–antibody reaction is finding application in other areas unrelated
to the blood typing of individuals. Most significantly, this approach
has been extended to the
1
detection of drugs in blood and urine. Antibodies that react with drugs do not naturally exist; how8
ever, 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–protein7complex acts as an antigen stimulating the animal to produce antibodies (see Figure 10–3). The recovered blood serum of the
1 to the drug.
animal will contain antibodies that are specific or nearly specific
HO
HO
ISBN 1-256-36593-9
B
U
O
O
NCH3
NCH3
HO
HO
Drug
Protein carrier
Drug antibodies
FIGURE 10–3
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
245
246
CHAPTER 10
Several immunological assay techniques are commercially available for detecting drugs
through an antigen–antibody reaction. One such technique, the enzyme-multiplied immunoassay
technique (EMIT), has gained widespread popularity among toxicologists because of its speed
and high sensitivity for detecting drugs in urine.
Enzyme-Multiplied Immunoassay Technique (EMIT)
A typical EMIT analysis begins by adding to a subject’s urine antibodies that bind to a particular
type or class of drug being looked for. This is followed by adding to the urine a chemically labeled
version of the drug. As shown in Figure 10–4, a competition will ensue between the labeled and
unlabeled drug (if it’s present in the subject’s urine) to bind with the antibody. If this competition
does occur in a person’s urine, it signifies
that the urine screen test was positive for the drug beG
ing tested. For example, to check someone’s urine for methadone, the analyst would add
O labeled methadone to the urine. Any methadone present in
methadone antibodies and chemically
the urine immediately competes R
with the labeled methadone to bind with the methadone antibodies. The quantity of chemically labeled methadone left uncombined is then measured, and this
D of methadone originally present in the urine.
value is related to the concentration
One of the most frequent uses
Oof EMIT in forensic laboratories has been for screening the
urine of suspected marijuana users. The primary pharmacologically active agent in marijuana is
tetrahydrocannabinol, or THC. ToN
facilitate the elimination of THC, the body converts it to a series of substances called metabolites
, that are more readily excreted. The major THC metabolite
found in urine is a substance called THC-9-carboxylic acid. Antibodies against this metabolite are
prepared for EMIT testing. Normally the urine of marijuana users contains a very small quantity
of THC-9-carboxylic acid (less than
J one-millionth of a gram); however, this level is readily
detected by EMIT.
E
The greatest problem with detecting marijuana in urine is interpretation. Although smoking
S of THC metabolite, it is difficult to determine when the inmarijuana will result in the detection
dividual actually used marijuana.SIn individuals who smoke marijuana frequently, detection is
possible within two to five days after the last use of the drug. However, some individuals may
I after the last use of marijuana.
yield positive results up to thirty days
Currently, thousands of individuals
C regularly submit to urinalysis tests for the presence of
drugs of abuse. These individuals include military personnel, transportation industry employees,
A and subjects requiring preemployment drug screening.
police and corrections personnel,
Immunoassay testing for drugs has proven quite suitable for handling the large volume of
Labeled drug
1
*
*
*
Antibody
*
*
*
Antibody
*
L
E*
I
G
H
1
8
7
1
B
U
No drug
present in
urine
*
*
*
*
*
Drug present
in urine
*
*
*
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
ISBN 1-256-36593-9
FIGURE 10–4
In the EMIT assay, a drug that may be present in a urine specimen will compete with
added labeled drugs for a limited number of antibody binding sites. The labeled
drugs are indicated by an asterisk. Once the competition for antibody sites is
completed, the number of remaining unbound labeled drug is proportional to the
drug’s concentration in urine.
FORENSIC SEROLOGY
247
specimens that must be rapidly analyzed for drug content on a daily basis. Testing laboratories
have access to many commercially prepared sera arising from animals being injected with any
one of a variety of drugs. A particular serum that has been added to a urine specimen is designed
to interact with opiates, cannabinoids, cocaine, amphetamines, phencyclidine, barbiturates,
methadone, or other drugs. A word of caution: immunoassay is only presumptive in nature, and
its result must be confirmed by additional testing. Specifically, the confirmation test of choice is
gas chromatography-mass spectrometry, which is described in more detail in Chapter 9.
Monoclonal Antibodies
As we have seen in the previous section, when an animal such as a rabbit or mouse is injected
with an antigen, the animal responds by producing antibodies G
designed to bind to the invading
antigen. However, the process of producing antibodies designed to respond to foreign antigens
O sites to which an antibody
is complex. For one, an antigen typically has structurally different
may bind. So when the animal is actively producing attack antibodies,
it produces a series of
R
different antibodies, all of which are designed to attack some particular site on the antigen of
D However, the disadvantage
interest. These antibodies are known as polyclonal antibodies.
of polyclonal antibodies is that an animal can produce antibodies
O that vary in composition
over time. As a result, different batches of polyclonals may vary in their specificity and their
N
ability to bind to a particular antigen site.
As the technologies associated with forensic science have ,grown in importance, a need has
developed, in some instances, to have access to antibodies that are more uniform in their composition and attack power than the traditional polyclonals. This is best accomplished by adopting a
process in which an animal will produce antibodies designed toJattack one and only one site on
an antigen. Such antibodies are known as monoclonal antibodies.
E How can such monoclonals
be produced? The process begins by injecting a mouse with the antigen of interest. In response,
S
the mouse’s spleen cells will produce antibodies to fight off the invading
antigen. The spleen cells
are removed from the animal and are fused to fast-growingSblood cancer cells to produce
hybridoma cells. The hybridoma cells are then allowed to multiply and are screened for their speI activity of interest are then
cific antibody activity. The hybridoma cells that bear the antibody
selected and cultured. The rapidly multiplying cancer cells linked
C to the selected antibody cells
produce identical monoclonal antibodies in a limitless supply (see Figure 10–5).
A forensic test kits with inMonoclonal antibodies are being incorporated into commercial
creasing frequency. Many immunoassay test kits for drugs of abuse are being formulated with
monoclonal antibodies. Also, a recently introduced test for seminal material that incorporates a
L
monoclonal antibody has found wide popularity in crime laboratories (see page 236).
E approved a monoclonal drug
As a side note, in 1999 the U.S. Food and Drug Administration
treatment for cancer. Rituxin is a nontoxic monoclonal antibody designed to attack and destroy
I
cancerous white blood cells containing an antigen designated as CD20. Other monoclonal drug
Gto fulfill their long-held expectreatments are in the pipeline. Monoclonals are finally beginning
tation as medicine’s version of the “magic bullet.”
H
polyclonal antibodies
Antibodies produced by injecting
animals with a specific antigen; a
series of antibodies are produced
responding to a variety of different
sites on the antigen
monoclonal antibodies
A collection of identical
antibodies that interact with a
single antigen site
hybridoma cells
Fused spleen and tumor cells; used
to produce identical monoclonal
antibodies in a limitless supply
1
Forensic Characterization of Bloodstains
8 dried blood: (1) Is it blood?
The criminalist must answer the following questions when examining
(2) From what species did the blood originate? (3) If the blood7is of human origin, how closely
can it be associated with a particular individual?
1
B
The determination of blood is best made by means of a preliminary color test. For many years,
U color test; however, because
the most commonly used test for this purpose was the benzidine
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Color Tests
benzidine has been identified as a known carcinogen, its use has generally been discontinued, and
the chemical phenolphthalein is usually substituted in its place (this test is also known as the
Kastle-Meyer color test).1 Both the benzidine and Kastle-Meyer color tests are based on the
1
S. Tobe et al., “Evaluation of Six Presumptive Tests for Blood, Their Specificity, Sensitivity, and Effect on High
Molecular-Weight DNA,” Journal of Forensic Sciences 52 (2007): 102–109.
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248
CHAPTER 10
Antigen
1. Inject mouse or rabbit
with antigen.
Antibodies
Spleen
cells
2. Remove spleenG
and isolate spleen
O
cells, which produce
antibodies to the R
antigen of interest.
Malignant
blood
cells
D
O
Spleen
cellN
,
J
E
S
S
I
C
A
L
E
I
G
H
3. Fuse spleen cells with
malignant cells, which
grow well in culture.
Hybridoma
cells
4. Grow hybrid cells and
isolate ones that produce
the antibody of interest.
5. Culture the hybrid
cells to create a virtually
limitless supply of
antibodies.
Monoclonal
antibodies
FIGURE 10–5
Steps required to produce monoclonal antibodies.
hemoglobin
A red blood cell protein that
transports oxygen in the
bloodstream; it is responsible for
the red color of blood
See a Color Test for Blood
www.mycrimekit.com
may turn Kastle-Meyer pink. These substances include potatoes and horseradish. However, it is
unlikely that such materials will 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 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 is indicative of blood.
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
ISBN 1-256-36593-9
WEBEXTRA 10.1
1
8
7 possesses peroxidase-like activity. Peroxidases are enzymes
observation that blood hemoglobin
that accelerate the oxidation of 1
several classes of organic compounds by peroxides. When a
bloodstain, phenolphthalein reagent, and hydrogen peroxide are mixed together, the blood’s
B
hemoglobin causes the formation of a deep pink color.
The Kastle-Meyer test is not U
a specific test for blood; some vegetable materials, for instance,
FORENSIC SEROLOGY
249
G
O
R
D
FIGURE 10–6
(a) A section of a linoleum floor photographed under normal
O light. This floor was
located in the residence of a missing person. (b) Same section of the floor shown in
part (a) after spraying with luminol. A circular pattern was N
revealed. Investigators
concluded that the circular blood pattern was left by the bottom
of a bucket carried
,
about during the cleaning up of the blood. A small clump of sponge, blood, and hair
was found near where this photograph was taken.
J
E
Luminol and Bluestar
S luminol test.2 Unlike the benAnother important presumptive identification test for blood is the
zidine and Kastle-Meyer tests, the reaction of luminol with bloodSproduces light rather than color.
By spraying luminol reagent onto a suspect item, investigators can quickly screen large areas for
I
bloodstains. The sprayed objects must be located in a darkened area while being viewed for the
C blue glow. A relatively new
emission of light (luminescence); any bloodstains produce a faint
product, Bluestar, is now available to be used in place of luminol (http://www.bluestar-forensic
A
.com). Bluestar is easy to mix in the field. Its reaction with blood can be observed readily withCourtesy North Carolina State Bureau of Investigation
out having to create complete darkness. The luminol and Bluestar tests are extremely sensitive—
capable of detecting bloodstains diluted to as little as 1 in 100,000.
L For this reason, spraying large
areas such as carpets, walls, flooring, or the interior of a vehicle may reveal blood traces or patE
terns that would have gone unnoticed under normal lighting conditions
(see Figure 10–6). It is
important to note that luminol and Bluestar do not interfere withI any subsequent DNA testing.3
Microcrystalline Tests
luminol
The most sensitive chemical test
that is capable of presumptively
detecting bloodstains diluted to as
little as 1 in 100,000; its reaction
with blood emits light and thus
requires the result to be observed
in a darkened area
G
The identification of blood can be made more specific if microcrystalline
tests are performed on
H
the material. Several tests are available; the two most popular ones are the Takayama and
Teichmann tests. Both of these depend on the addition of specific chemicals to the blood so that
characteristic crystals with hemoglobin derivatives will form. Crystal
tests are far less sensitive
1
than color tests for blood identification and are more susceptible to interference from contami8
nants that may be present in the stain.
7
1
Once the stain has been characterized as blood, the serologist determines whether the stain is of
human or animal origin. For this purpose, the standard test usedBis the precipitin test. Precipitin
tests are based on the fact that when animals (usually rabbits) are injected with human blood,
U
antibodies form that react with the invading human blood to neutralize its presence. The investiISBN 1-256-36593-9
Precipitin Test
gator can recover these antibodies by bleeding the animal and isolating the blood serum. This
precipitin
An antibody that reacts with its
corresponding antigen to form a
precipitate
2
The luminol reagent is prepared by mixing 0.1 grams 3-amino-phthalhydrazide and 5.0 grams sodium carbonate in
100 milliliters distilled water. Before use, 0.7 grams sodium perborate is added to the solution.
3
A. M. Gross et al., “The Effect of Luminol on Presumptive Tests and DNA Analysis Using the Polymerase Chain
Reaction,” Journal of Forensic Sciences 44 (1999): 837.
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250
CHAPTER 10
serum 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. Currently, antiserums
are commercially available for humans and for a variety of commonly encountered animals—for
example, dogs, cats, and deer.
A number of 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, as indicated by the formation of a cloudy
ring or band at the interface of the two liquids (see Figure 10–7).
Another version of the precipitin test, called gel diffusion, takes advantage of the fact that
Gmove toward one another on an agar gel–coated plate. The
antibodies and antigens diffuse or
extracted bloodstain and the human
Oantiserum are placed in separate holes opposite each other on
the gel. If the blood is of human origin, a line of precipitation will form where the antigens and
R and antibodies can be induced to move toward one anantibodies meet. Similarly, the antigens
other under the influence of an electrical
D field. In the electrophoretic method (see pages 131–132),
an electrical potential is applied to the gel medium; a specific antigen–antibody reaction is deO
noted by a line of precipitation formed between the hole containing the blood extract and the hole
containing the human antiserum (see
N Figure 10–8).
The precipitin test is very sensitive and requires only a small amount of blood for testing.
,
Human bloodstains dried for 10 to 15 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,
J human bloodstains diluted by washing in water and left with
only a faint color may still yield a positive precipitin reaction (see Figure 10–9).
E that the bloodstain is of human origin, an effort must be
Once it has been determined
made to associate or disassociateSthe stain with a particular individual. Until the mid-1990s,
routine characterization of bloodstains included the determination of A-B-O types; however,
S or typing has relegated this subject to one of historical
the widespread use of DNA profiling
interest only.
I
C
A
L
E
I
G
Withdrawing blood
from human vein H
1
8
7
1
Human
B blood
gives a precipitin
bandU
with sensitized
Rabbit serum
sensitized to
human blood is
removed from rabbit
Human
blood
Rabbit
serum
FIGURE 10–7
The precipitin test.
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
ISBN 1-256-36593-9
rabbit serum
Blood injected
into rabbit
FORENSIC SEROLOGY
−
Semen extract
and anti-p30 are
added to their
respective wells
251
+
Antigen and antibody
move toward
each other
Formation of a visible
precipitation line midway
between the wells shows
the presence of p30 in the
FIGURE 10–8
G the influence of an
Antigens and antibodies moving toward one another under
electrical potential.
O
R
D
O
N
,
J
E
S
S
FIGURE 10–9
I
Results of the precipitin test of dilutions of human serumC
up to 1 in 4,096 against a
human antiserum. A reaction is visible for blood dilutions up to 1 in 256.
Courtesy Millipore Corp., Biomedical, Action, Mass.
A
Principles of Heredity
L
All of the antigens and polymorphic enzymes and proteins thatE
have been described in previous
sections are genetically controlled traits. That is, they are inherited
I from parents and become a
permanent feature of a person’s biological makeup from the moment he or she is conceived.
G us with a picture of how one
Determining the identity of these traits, then, not only provides
individual compares to or differs from another, but gives us anH
insight into the basic biological
gene
1
Genes and Chromosomes
8
Hereditary material is transmitted via microscopic units called genes. The gene is the basic unit
7
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
1 nature and growth of virtually
every body structure.
B
The genes are positioned on chromosomes, threadlike bodies that appear in the nucleus of
every body cell. See Figure 10–10. All nucleated human cells contain
U 46 chromosomes, mated in
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ISBN 1-256-36593-9
substances that determine our overall makeup as human beings and the mechanism by which
those substances are transmitted from one generation to the next.
23 pairs. The only exceptions are the human reproductive cells, the egg and sperm, which contain
only 23 unmated chromosomes. During fertilization, a sperm and egg combine so that each contributes chromosomes to form the new cell (zygote). Hence, the new individual begins life properly with 23 mated chromosome pairs. Because the genes are positioned on the chromosomes, the
new individual inherits genetic material from each parent.
Actually, two dissimilar chromosomes are involved in the determination of sex. The egg cell
always contains a long chromosome known as the X chromosome; but the sperm cell may
A unit of inheritance consisting of
a DNA segment located on a
chromosome
WEBEXTRA 10.2
Learn about the Chromosomes
Present in Our Cells
chromosome
A rodlike structure in the cell
nucleus, along which the genes are
located; it is composed of DNA
surrounded by other material,
mainly proteins
egg
The female reproductive cell
sperm
The male reproductive cell
zygote
The cell arising from the union of
an egg and a sperm cell
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252
CHAPTER 10
FIGURE 10–10
Computer-enhanced
photomicrograph image of
human chromosomes.
Courtesy Alfred Pasieka, Science Photo
Library, Photo Researchers, Inc.
G
O
R
D
O
N
,
X chromosome
The female sex chromosome
Y chromosome
The male sex chromosome
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; for example, the
genes determining the blood types
A and B are alleles
homozygous
Having two identical allelic genes
on two corresponding positions of
a pair of chromosomes
heterozygous
J
E
S
S known as the Y chromosome, or a long X chromosome.
contain either a short chromosome,
When an X-carrying sperm fertilizes
I an egg, the new cell is XX and develops into a female. A Ycarrying sperm produces an XY fertilized egg and develops into a male. Because the sperm cell
Cthe chromosome pair, we can say that the father biologically
ultimately determines the nature of
determines the sex of the child. A
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
L
are similarly positioned on the chromosomes inherited from the mother and father. Thus, a
gene for eye color on the mother’s
E chromosome will be aligned with a gene for eye color on
the corresponding chromosome inherited from the father. Alternative forms of genes that inI
fluence a given characteristic and are aligned with one another on a chromosome pair are
G
known as alleles.
Another simple example of allele
H genes in humans is that of blood types belonging to the
A-B-O system. Inheritance of the A-B-O type is best described by a theory that uses three genes
designated A, B, and O. A gene pair made up of two similar genes—for example, AA and BB—
is said to be homozygous; a gene 1
pair made up of two different genes—AO, for example—is said
to be heterozygous. If the chromosome inherited from the father carries the A gene and the
chromosome inherited from the 8mother carries the same gene, the offspring would have an
AA combination. Similarly, if one7chromosome contains the A gene and the other has the O gene,
the genetic makeup of the offspring would be AO.
Having two different allelic genes
on two corresponding positions of
a pair of chromosomes
1
B When an individual inherits two similar genes from his
or her parents, there is no problem in determining the blood type of that person. Hence, an AA
U a BB type B, and an OO type O. However, when two differcombination will always be type A,
WEBEXTRA 10.3
ent genes are inherited, one gene will be dominant. It can be said that the A and B genes are
dominant and that the O gene is always recessive—that is, its characteristics remain hidden. For
instance, with an AO combination, A is always dominant over O, and the individual will be 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. Hence, the combination OO is typed simply as O.
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ISBN 1-256-36593-9
Learn about the Structure of Our
Genes
DOMINANT AND RECESSIVE GENES
FORENSIC SEROLOGY
GENOTYPES AND PHENOTYPES A pair of allele genes together constitutes the genotype of the
individual. However, no laboratory test can determine an individual’s A-B-O genotype. For
example, a person’s outward characteristic, or phenotype, may be type A, but this does not tell
us whether his or her genotype is AA or AO. The genotype can be determined only by studying
the family history of the individual. If the genotypes of both parents are known, that of their
possible offspring can be forecast.
An easy way to figure this out is to construct a Punnett square. To do this, write along a
horizontal line the two genes of the male parent, and in the vertical column write the two
kinds of female genes present, as shown. In our example, we assume the male parent is type
O and therefore has to be an OO genotype; the female parent is type AB and can be only an
AB genotype:
Father’s
genotype
O
O
A
Mother’s
genotype
B
253
genotype
The particular combination of
genes present in the cells of an
individual
phenotype
The physical manifestation of a
genetic trait such as shape, color,
and blood type
G
O
R
D
O
N
,
Next, write in each box the corresponding gene contributed from the female and then from
J
the male. The squares will contain all the possible genotype combinations
that the parents can
produce in their offspring:
E
O
O
A
AO
AO
B
BO
BO
S
S
I
C
A
Hence, in this case, 50 percent of the offspring are likely to be AO and the other 50 percent
L
BO. These are the only genotypes possible from this combination.
Because O is recessive,
50 percent of the offspring will probably be type A and 50 percent
type
B. From this example,
E
we can see that no blood group gene can appear in a child unless it is present in at least one
I
of the parents.
ISBN 1-256-36593-9
Paternity Testing
WEBEXTRA 10.4
See How Genes Position Themselves on a Chromosome Pair
www.mycrimekit.com
WEBEXTRA 10.5
See How Genes Define Our Genetic
Makeup
www.mycrimekit.com
G
H
Although the genotyping of blood factors has useful applications for studying the transmission of
blood characteristics from one generation to the next, it has no direct relevance to criminal in1
vestigations. It does, however, have important implications in disputed-paternity
cases, which are
normally encountered in civil, not criminal, courts.
8
Many cases of disputed paternity can be resolved when the suspected parents and the off7
spring are related according to their blood group systems. For instance,
in the previous example,
had the child been type AB, the suspected father would have been
cleared.
A type O father and a
1
type AB mother cannot have a type AB child. On the other hand, if the child had been type A or
B the father; this does not mean
type B, the most that could be said is that the suspect may have been
that he is the father, just that he is not excluded based on blood
Utyping. Obviously, many other
males also have type O blood. Of course, the more blood group systems that are tested, the better the chances of excluding an innocent male from involvement. Conversely, if no discrepancies
are found between offspring and suspect father, the more certain one can be that the suspect is
indeed the father. In fact, routine paternity testing involves characterizing blood factors other than
A-B-O. Currently, paternity testing laboratories have implemented DNA test procedures that can
raise the odds of establishing paternity beyond 99 percent.
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254
CHAPTER 10
Forensic Characterization of Semen
Many cases received in a forensic laboratory involve sexual offenses, making it necessary to
examine exhibits for the presence of seminal stains.
The normal male releases 2.5 to 6 milliliters of seminal fluid during an ejaculation. Each
milliliter contains 100 million or more spermatozoa, the male reproductive cells. Forensic
examination of articles for seminal stains can actually be considered a two-step process. First,
before any tests can be conducted, the stain must be located. Considering the number and soiled
condition of outer garments, undergarments, and possible bedclothing submitted for examination,
this may prove to be an arduous task. Once located, the stain will have to be subjected to tests that
will prove its identity; it may even be tested for the blood type of the individual from whom it
G
originated.
O
R on a fabric because they exhibit a stiff, crusty appearance.
Often, seminal stains are readily visible
However, reliance on such appearance
D for locating the stain is at best unreliable and is useful only
when the stain is present in a rather obvious area. Certainly, if the fabric has been washed or
contains only minute quantities ofO
semen, visual examination of the article offers little chance of
detecting the stain. The best way N
to locate and characterize a seminal stain is to perform the acid
phosphatase color test.
,
Testing for Seminal Stains
acid phosphatase
An enzyme found in high
concentration in semen
Acid phosphatase is an enzyme that is secreted by the prostate gland
into seminal fluid. Its concentrations in seminal fluid are up to 400 times greater than those found
J can easily be detected when it comes in contact with an acidic
in any other body fluid. Its presence
solution of sodium alpha naphthylphosphate
and Fast Blue B dye. Also, 4-methyl umbelliferyl
E
phosphate (MUP) fluoresces under UV light when it comes in contact with acid phosphatase.
S
The utility of the acid phosphatase
test is apparent when it becomes necessary to search
numerous garments or large fabric
areas
for
seminal stains. If a filter paper is simply moistened
S
with water and rubbed lightly over the suspect area, acid phosphatase, if present, is transferred to
I or two of the sodium alpha naphthylphosphate and Fast Blue
the filter paper. Then, when a drop
B solution are placed on the paper,C
the appearance of a purple color indicates the acid phosphatase
enzyme. In this manner, any fabric or surface can be systematically searched for seminal stains.
A
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 under investigation can be pressed against a suitably sized piece of
L
moistened filter paper. The paper is then sprayed with MUP solution. Semen stains appear as
E light. A negative reaction can be interpreted as meaning the
strongly fluorescent areas under UV
absence of semen. Although some
I vegetable and fruit juices (such as cauliflower and watermelon), fungi, contraceptive creams, and vaginal secretions give a positive response to the acid
phosphatase test, none of these G
substances normally reacts with the speed of seminal fluid.
A reaction time of less than 30 seconds
H is considered a strong indication of the presence of semen.
ACID PHOSPHATASE TEST
Semen can be unequivocally identified by the presence of spermatozoa. When spermatozoa are located through a microscope examination, the stain
is definitely identified as having 1
been derived from semen. Spermatozoa are slender, elongated
structures 50–70 microns long, each
8 with a head and a thin flagellate tail (see Figure 10–11). The
criminalist can normally locate them by immersing the stained material in a small volume of
7
water. Rapid stirring of the liquid transfers a small percentage of the spermatozoa present into the
water. A drop of the water is dried
1 onto a microscope slide, then stained and examined under a
compound microscope at a magnification of approximately 400 .4
B
Considering the extremely large number of spermatozoa found in seminal fluid, one would
U be very good; however, this is not always true. One reathink the chance of locating one would
son is that spermatozoa are bound tightly to cloth materials.5 Also, spermatozoa are extremely
MICROSCOPIC EXAMINATION OF SEMEN
J. P. Allery et al., “Cytological Detection of Spermatozoa: Comparison of Three Staining Methods,” Journal of
Forensic Sciences 46 (2001): 349.
5
In one study, only a maximum of 4 sperm cells out of 1,000 could be extracted from a cotton patch and observed under
the microscope. Edwin Jones (Ventura County Sheriff’s Department, Ventura, Calif.), personal communication.
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ISBN 1-256-36593-9
4
FORENSIC SEROLOGY
255
G
O
R
D
O
N
,
FIGURE 10–11
Photomicrograph of human spermatozoa (300 ).
Courtesy John Walsh, Photo Researchers, Inc.
−
Antigen and antibody
are added to their
respective wells
+
Antigen and antibody
move toward
each other
J
E
S
S
I
C
A
Antigen and antibody
have formed a visible
precipitin line in the
gel between the wells
L
E
I
FIGURE 10–12
G
H
brittle when dry and easily disintegrate if the stain is washed or when the stain is rubbed against
another object, as can happen 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 con1
dition known as oligospermia, or they may involve individuals who have no spermatozoa at all
8
in their seminal fluid (aspermia). Significantly, aspermatic individuals
are increasing in numbers
because of the growing popularity of vasectomies.
7
Forensic analysts often must
1 examine stains or swabs that
they suspect contain semen (because of the presence of acid phosphatase) but that yield no deB presence of semen? The solutectable spermatozoa. How, then, can one unequivocally prove the
tion to this problem came with the discovery in the 1970s of U
a protein called p30 or prostate
specific antigen (PSA). Under the analytical conditions employed in forensic laboratories, p30 is
unique to seminal plasma.
When p30 is isolated and injected into a rabbit, it stimulates the production of polyclonal
antibodies (anti-p30). The sera collected from these immunized rabbits can then be used to test
suspected semen stains. As shown in Figure 10–12, 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,
ISBN 1-256-36593-9
PROSTATE-SPECIFIC ANTIGEN (PSA)
oligospermia
An abnormally low sperm count
aspermia
The absence of sperm; sterility
in males
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256
CHAPTER 10
Human PSA
(antigen) extracted
from a suspect
stain
Reaction
zone
Positive
test
Blue
dye
Mobile monoclonal
PSA antibody
attached to a dye
Mobile
G antigen–
antibody complex
O toward
migrates
reaction zone
Polyclonal
PSA antibody
Antibody–
antigen–
antibody
sandwich
seen as a
blue line
R
D
FIGURE 10–13
O
An antibody–antigen–antibodyNsandwich or complex is seen as a colored band. This
signifies the presence of PSA in the extract of a stain and positively identifies human
,
semen.
the antigens and antibodies move toward each other. The formation of a visible line midway
J
between the two wells shows the presence of p30 in the stain and proves that the stain was seminal
in nature.
E
A more elegant approach to identifying PSA (p30) involves placing an extract of a questioned
S
sample on a porous membrane in the presence of a monoclonal PSA antibody that is linked to a dye.
If PSA is present in the extract, aSPSA antigen–monoclonal PSA antibody complex forms. This
complex then migrates along the membrane,
where it interacts with a polyclonal PSA antibody emI
bedded in the membrane. The antibody–antigen–antibody “sandwich” that forms will be apparent
C Figure 10–13). This monoclonal antibody technique is about
by the presence of a colored line (see
a hundred times more sensitive forA
detecting PSA than the one described in the previous paragraph.6
Once the material under examination is proven to be semen, the next task is to attempt to
associate the semen as closely as possible with a single individual. As we will learn in Chapter 11,
forensic scientists can link seminal
L material to one individual with DNA technology. Just as
important is the knowledge that this technology can exonerate many of those wrongfully accused
E
of sexual assault.
I
G
Preservation
H
Collection and
of Rape Evidence
Seminal constituents on a rape victim
1 are important evidence that sexual intercourse has taken
place, but their absence does not necessarily mean that a rape did not occur. Physical injuries such
8 that a violent assault did take place. Furthermore, the forceas bruises or bleeding tend to confirm
ful physical contact between victim
7 and assailant may result in a transfer of physical evidence—
blood, semen, hairs, and fibers. The presence of such physical evidence will help forge a vital link
1
in the chain of circumstances surrounding
a sexual crime.
B
U
To protect this kind of evidence, all the outer and undergarments from the involved parties should
Collection and Handling
6
J. Kearsey, H. Louie, and H. Poon, “Validation Study of the Onestep ABAcard® PSA Test Kit for RCMP Casework,”
Canadian Society of Forensic Science Journal 34 (2001): 63; S. J. Denison, E. M. Lopes, L. D’Costa, and J. C. Newman,
“Positive Prostate-Specific Antigen (PSA) Results in Semen-Free Samples,” Canadian Society of Forensic Science
Journal 37 (2004): 197.
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ISBN 1-256-36593-9
be carefully removed and packaged separately in paper (not plastic) bags. Place a clean bedsheet
FORENSIC SEROLOGY
on the floor and lay a clean paper sheet over it. The victim must remove her shoes before standing on the paper. Have the person disrobe while standing on the paper in order to collect any loose
foreign material falling from the clothing. Collect each piece of clothing as it is removed and
place the items in separate paper bags to avoid cross-contamination of physical evidence. Carefully fold the paper sheet so that all foreign materials are contained inside.
If it is deemed 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 through the 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 with an unstained piece as a substrate control.
In the laboratory, analysts try to link seminal material toGa donor(s) using DNA typing.
Because an individual may transfer his or her DNA types to a stain
O through perspiration, investigators must handle stained articles with care, minimizing direct personal contact. The evidence
collector must wear disposable latex gloves when such evidenceRmust be touched.
The rape victim must undergo a medical examination as soon as
Dpossible after the assault. At this
time, the appropriate items of physical evidence are collected by trained personnel. Evidence collecO
tors should have an evidence-collection kit from the local crime laboratory (see Figures 10–14,
10–15, and 10–16).
N
The following items of physical evidence are to be collected:
,
1. Pubic combings. Place a paper towel under the buttocks and comb the pubic area for loose
or foreign hairs.
J
2. Pubic hair standard/reference samples. Cut 15 to 20 full-length
hairs from the pubic area
at the skin line.
E
3. External genital dry-skin areas. Swab with at least one dry swab and one moistened swab.
S
S
I
FIGURE 10–14
C
Victim rape collection kit
A
showing the kit envelope, kit
instructions, medical history
and assault information
L
forms, and foreign-materials
collection bag.
E
Courtesy Tri-Tech, Inc., Southport,
IN.C., www.tritechusa.com
G
H
ISBN 1-256-36593-9
1
8
7
1
B
U
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257
258
CHAPTER 10
FIGURE 10–15
Victim rape collection kit
showing collection bags for
outer clothing, underpants,
debris, pubic hair combings,
pubic hair standard/reference
samples, vaginal swabs, and
rectal swabs.
Courtesy Tri-Tech, Inc., Southport, N.C.,
www.tritechusa.com
G
O
R
D
O
N
,
J
E
S
S
I
C
A
FIGURE 10–16
Victim collection rape kit
showing collection bags for
oral swabs and smear, pulled
head hair standard/reference,
known saliva sample, known L
blood samples, and
E
anatomical drawings.
Courtesy Tri-Tech, Inc., Southport, N.C.,
www.tritechusa.com
I
G
H
1
8
7
1
B
U
ISBN 1-256-36593-9
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FORENSIC SEROLOGY
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.
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 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 buccal area and gum line. Using both swabs, prepare one smear
G
slide. Allow both swabs and the smear to air-dry before packaging.
8. Head hairs. Cut at skin line a minimum of five full-length
Ohairs from each of the following scalp locations: center, front, back, left side, and right side. It is recommended that a total
of at least 50 hairs be cut and submitted to the laboratory. R
9. Blood sample. Collect at least 20 milliliters in a vacuumD
tube containing the preservative
EDTA. The blood sample can be used for DNA typing as well as for toxicological analysis
O
if required.
10. Fingernail scrapings. Scrape the undersurface of the nails
Nwith 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 the purpose of
conducting a drug toxicological analysis for Rohypnol, GHB,
J and other substances associated with drug-facilitated sexual assaults (see pages 200–201).
E
Often during the investigation of a sexual assault, the victim reports that a perpetrator
S As we will learn in the next
engaged in biting, sucking, or licking of areas of the victim’s body.
chapter, the tremendous sensitivity associated with DNA technology
offers investigators the
S
opportunity to identify a perpetuator’s DNA types from saliva residues collected off the skin. The
I
most efficient way to recover saliva residues from the skin is to first swab the suspect area with
C A second, dry swab is then
a rotating motion using a cotton swab moistened with distilled water.
rotated over the skin to recover the moist remains on the skin’s surface from the wet swab. The
A
swabs are air-dried and packaged together as a single sample.7
If a suspect is apprehended, the following items are routinely collected:
1.
2.
3.
4.
5.
Lat the time of assault.
All clothing and any other items believed to have been worn
Pubic hair combings.
E
Pulled head and pubic hair standard/reference samples.
I
Penile swab within twenty-four hours of assault when appropriate
to case history.
A blood sample or buccal swab (see page 287) for DNA-typing
purposes.
G
ISBN 1-256-36593-9
The advent of DNA profiling has forced investigators toH
rethink what items are evidential with respect to a sexual assault. As we will learn in Chapter 11, DNA levels in the range
of one-billionth of a gram are now routinely characterized in crime laboratories. In the past,
1 who was suspected of being
scant attention was paid to the underwear recovered from a male
involved in a sexual assault. From a practical point of view,
8 the presence of seminal
constituents on a man’s underwear had little or no investigative value. Today, the high sensi7 Experience now tells us that
tivity of DNA analysis has created new areas of investigation.
it is possible to establish a link between a victim and her assailant
by analyzing biological
1
material recovered from the interior front surface of a male suspect’s underwear. This is
B presence of a suspect’s DNA
especially important when investigations have failed to yield the
on exhibits recovered from the victim.
U
ANALYZING SEMINAL CONSTITUENTS The persistence of seminal constituents in the vagina
may become a factor when trying to ascertain the time of an alleged sexual attack. Although the
7
D. Sweet et al., “An Improved Method to Recover Saliva from Human Skin: The Double Swab Technique,” Journal
of Forensic Sciences 42 (1997): 320.
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260
CHAPTER 10
forensic brief
> > > > > > > > > > > > >> > > >
underwear revealed a female DNA profile matching
that of the victim. The added bonus in this case was
finding male DNA on the same underwear that
matched that of the consensual partner.
A common mode of DNA transfer occurs when skin
cells from the walls of the 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 aptly illustrated in a
case in which the female victim of a rape had consensual sexual intercourse with a male partner prior
to being assaulted by a different male. DNA extracted from the inside front area of the suspect’s
WEBEXTRA 10.6
Step into the Role of the First
Responding Officer at a Sexual
Assault Scene
www.mycrimekit.com
WEBEXTRA 10.7
Assume the Duties of an EvidenceCollection Technician at a Sexual
Assault Scene
www.mycrimekit.com
Virtual Forensics Lab
Conduct an Analysis
of a Bloodstain
Source: 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.
G
O
R
D
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N cavity provides evidence of intercourse, important inforpresence of spermatozoa in the vaginal
mation regarding the time of sexual
, activity can be obtained from the knowledge that motile or
living sperm generally survive up to four to six hours in the vaginal cavity of a living person.
However, a successful search for motile sperm requires a microscopic examination of a vaginal
smear immediately after it is taken
Jfrom the victim.
A more extensive examination of vaginal collections is later made at a forensic laboratory.
E
Nonmotile sperm may be found in a living female for up to three days after intercourse and occasionally up to six days later. However,
intact sperm (sperm with tails) are not normally found
S
16 hours after intercourse but have been found as late as 72 hours after intercourse. The likeliS
hood of finding seminal acid phosphatase in the vaginal cavity markedly decreases with time
following intercourse, with littleI chance of identifying this substance 48 hours after intercourse.8 Hence, with the possibility
C of the prolonged persistence of both spermatozoa and acid
phosphatase in the vaginal cavity after intercourse, investigators should determine when and if
A before the sexual assault. This information will be usevoluntary sexual activity last occurred
ful for evaluating the significance of finding these seminal constituents in the female victim.
Blood or buccal swabs for DNA analysis are to be taken from any consensual partner having
L before the assault.
sex with the victim within 72 hours
Another significant indicatorE
of recent sexual activity is p30. This semen marker normally is
not detected in the vaginal cavity beyond 24 hours following intercourse.9
I
G
H
To perform a virtual lab analysis
of a bloodstain, go to www
.pearsoncustom.com/us/vlm/
1
8
7
1
B
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Anne Davies and Elizabeth Wilson, “The Persistence of Seminal Constituents in the Human Vagina,” Forensic Science 3 (1974): 45.
9
J. Kearsey, H. Louie, and H. Poon, “Validation Study of the Onestep ABAcard® PSA Test Kit for RCMP Casework,”
Canadian Society of Forensic Science Journal 34 (2001): 63.
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
ISBN 1-256-36593-9
8
FORENSIC SEROLOGY
ISBN 1-256-36593-9
chapter summary
261
>>>>>>>>>>>
The term serology describes a broad scope of laboratory tests
that use specific antigen and serum antibody reactions. An antibody reacts or agglutinates only with its specific antigen. The
identity of each of the four A-B-O blood groups can be established by testing the blood with anti-A and anti-B sera. The
concept of specific antigen–antibody reactions has been applied to immunoassay techniques for detecting drugs of abuse
in blood and urine. When an animal is injected with an antigen, its body produces a series of different antibodies, all of
which are designed to attack some particular site on the antigen of interest. This collection of antibodies is known as
polyclonal antibodies. Alternately, a more uniform and specific collection of antibodies designed to combine with a single antigen site can be manufactured. Such antibodies are
known as monoclonals.
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 of human
origin, how closely can it be associated to a particular individual? The determination of blood is best made by means of
a preliminary color test. A positive result from the KastleMeyer color test is highly indicative of blood. Alternatively,
the luminol and Bluestar tests are used to search out trace
G amounts of blood located at crime scenes. The precipitin test
antisera normally derived from rabbits that have been inO uses
jected with the blood of a known animal to determine the
R species origin of a questioned bloodstain. Before the advent of
D DNA typing, bloodstains were linked to a source by A-B-O
typing. This approach has now been supplanted by the newer
O DNA technology.
N Many cases sent to a forensic laboratory involve sexual
offenses, making it necessary to examine exhibits for the pres, ence of seminal stains. The best way to locate and characterize a seminal stain is to perform the acid phosphatase color
test. Semen can be unequivocally identified by the presence of
J either spermatozoa or p30, a protein unique to seminal plasma.
E Forensic scientists can link seminal material to an individual
DNA typing. The rape victim must undergo a medical exS by
amination as soon as possible after the assault. At that time
S clothing, hairs, and vaginal and rectal swabs can be collected
subsequent laboratory examination. If a suspect is appreI for
hended within 24 hours of the assault, it may be possible to deC tect the victim’s DNA on the male’s underwear or on a penile
A swab of the suspect.
review questions
L
E
I 9.
G
H 10.
1. Karl Landsteiner discovered that blood can be classified
by its ___________.
2. True or False: No two individuals, except for identical
twins, can be expected to have the same combination of
blood types or antigens. ___________
3. ___________ is the fluid portion of unclotted blood.
4. The liquid that separates from the blood when a clot is
formed is called the ___________.
5. ___________ transport oxygen from the lungs to the
body tissues and carry carbon dioxide back to the lungs.
6. On the surface of red blood cells are chemical substances
called ___________, which impart blood type characteristics to the cells.
7. Type A individuals have ___________ antigens on the
surface of their red blood cells.
8. Type O individuals have (both A and B, neither A nor B)
antigens on their red blood cells.
11.
1
8 12.
7 13.
1
B 14.
U
15.
16.
17.
The presence or absence of the ___________
and ___________ antigens on the red blood cells determines a person’s blood type in the A-B-O system.
The D antigen is also known as the ___________ antigen.
Serum contains proteins known as ___________, which
destroy or inactivate antigens.
An antibody reacts with (any, only a specific) antigen.
True or False: Agglutination describes the clumping together of red blood cells by the action of an antibody.
___________
Type B blood contains ___________ antigens and anti___________ antibodies.
Type AB blood has (both anti-A and anti-B, neither antiA nor anti-B) antigens.
A drug–protein complex can be injected into an animal
to form specific ___________ for that drug.
The term ___________ describes the study of
antigen–antibody reactions.
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
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CHAPTER 10
18. Type AB blood (is, is not) agglutinated by both anti-A
and anti-B serum.
33. All nucleated cells in the human body, except the reproductive cells, have ___________ pairs of chromosomes.
19. Type B red blood cells agglutinate when added to type
(A, B) blood.
34. The sex of an offspring is always determined by the
(mother, father).
20. Type A red blood cells agglutinate when added to type
(AB, O) blood.
35. Genes that influence a given characteristic and are
aligned with one another on a chromosome pair are
known as ___________.
36. When a pair of allelic genes is identical, the genes are
said to be (homozygous, heterozygous).
37. A (phenotype, genotype) is an observable characteristic
G of an individual.
21. An immunological assay technique used to detect the
presence of minute quantities of drugs in blood and urine
is ___________.
22. The distribution of type A blood in the United States is
approximately (42, 15) percent.
23. The distribution of type AB blood in the United States is
approximately (12, 3) percent.
24. (All, Most) blood hemoglobin has peroxidase-like
activity.
25. For many years, the most commonly used color test for
identifying blood was the ___________ color test.
26. ___________ reagent reacts with blood, causing it to
luminesce.
27. Blood can be characterized as being of human origin by
the ___________ test.
28. Antigens and antibodies (can, cannot) be induced to
move toward each other under the influence of an electrical field.
29. Antibodies designed to interact with a specific antigen
site are (monoclonal, polyclonal).
30. True or False: Hybridoma cells are used to produce
antigens designed to attack one and only one site on an
antibody. ___________
31. The basic unit of heredity is the ___________.
32. Genes are positioned on threadlike bodies called
___________.
application and critical
O38.
R
39.
D
O40.
N
, 41.
42.
J
E43.
S44.
S
I 45.
C
46.
A47.
The combination of genes present in the cells of an individual is called the ___________.
A gene (will, will not) appear in a child when it is present in one of the parents.
A type B individual may have the genotype
___________ or the genotype ___________.
A type AB mother and type AB father will have offspring
of what possible genotypes? ___________
A type AB mother and type AB father will have offspring
of what possible phenotypes? ___________
The ___________ color test is used to locate and characterize seminal stains.
Semen is unequivocally identified by the microscopic
appearance of ___________.
Males with a low sperm count have a condition known
as (oligospermia, aspermia).
The protein ___________ is unique to seminal plasma.
True or False: DNA may be transferred to an object
through the medium of perspiration. ___________
L48. True or False: Seminal constituents may remain in the
vagina for up to six days after intercourse. ___________
E
I
G
H
thinking
1
8
7
1 2.
B
U
in his possession. Three minutes after completion of the
test, the blanket shows a positive reaction. What test
did Scott choose and what was his conclusion? Explain
your answer.
Criminalist Cathy Richards is collecting evidence from
the victim of a sexual assault. She places a sheet on the
floor, asks the victim to disrobe, and places the clothing
in a paper bag. After collecting pubic combings and
pubic hair samples, she takes two vaginal swabs, which
she allows to air-dry before packaging. Finally, Cathy
collects blood, urine, and scalp hair samples from the
victim. What mistakes, if any, did she make in collecting
this evidence?
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
ISBN 1-256-36593-9
1. Police investigating the scene of a sexual assault recover a large blanket that they believe may contain
useful physical evidence. They take it to the laboratory of forensic serologist Scott Alden, asking him to
test it for the presence of semen. Noticing faint pink
stains on the blanket, Scott asks the investigating detective if he is aware of anything that might recently
have been spilled on the blanket. The detective reports that an overturned bowl of grapes and watermelon was found at the scene, as well as a broken
glass that had contained wine. After the detective departs, Scott chooses and administers what he considers the best test for analyzing the piece of evidence
FORENSIC SEROLOGY
further references
Jones, E. L., Jr., “The Identification and Individualization
of Semen Stains,” in R. Saferstein, ed., Forensic Science
Handbook, vol. 2, 2nd ed. Upper Saddle River, N.J.:
Prentice Hall, 2005.
Virkler, K., and I. K. Lednev, “Analysis of Body Fluids
for Forensic Purposes: From Laboratory Testing to NonDestructive Rapid Confirmatory Identification at a Crime
Scene,” Forensic Science International 188 (2009): 1.
Shaler, R. C., “Modern Forensic Biology,” in R. Saferstein,
ed., Forensic Science Handbook, vol. 1, 2nd ed. Upper
Saddle River, N.J.: Prentice Hall, 2002.
G
O
R
D
O
N
,
J
E
S
S
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A
L
E
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263
Because of permissions issues, some material (e.g., photographs) has been removed from this chapter, though reference to it
may occur in the text. The omitted content was intentionally deleted and is not needed to meet the University’s requirements for
this course.
headline news
O. J. Simpson—A Mountain of Evidence
On June 12, 1994, police arrived at the
G Nicole Simpson only to view a
home of
horrificOscene. The bodies of O. J. Simpson’s
Rwife and her friend Ron Goldman were found on
estranged
the pathDleading to the front door of Nicole’s home. Both
bodies O
were covered in blood and had suffered deep
knife wounds.
Nicole’s head was nearly severed from her
N
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. Blood drops were on
J
O. J.’s driveway and in the foyer of his home.
E
A blood-soaked sock was located in O. J. Simpson’s
S
bedroom,
and a bloodstained glove rested outside
S
his residence.
I As DNA was extracted and profiled from each
Cbloodstained article, a picture emerged that
A seemed to irrefutably link Simpson to the
murders. A trail of DNA leaving the crime scene
L was consistent with O. J.’s profile, as was the
E DNA found entering Simpson’s home.
Simpson’s DNA profile was found in the
I
Bronco along with that of both victims. The glove
G
contained the DNA profiles of Nicole and Ron, and the sock had
H
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 was1tinged with dishonest police planting evidence.
8
The strategy worked. O. J. Simpson was acquitted of murder.
7
1
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U
ISBN 1-256-36593-9
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
>>>>>>>>>>>>
chapter
11
DNA: the indispensable
forensic science tool
Learning Objectives
G
O
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D
O
N
,
J
E
S
After studying this chapter you should
be able to:
S how they are
• Name the parts of a nucleotide and explain
I
linked together to form DNA
C it relates to the
• Understand the concept of base pairing as
double-helix structure of DNA
A
• Contrast DNA strands that code for the production of proteins
with strands that contain repeating base sequences
L
• Explain the technology of polymerase chain
E reaction (PCR) and
how it applies to forensic DNA typing
•
•
I
Understand the structure of an STR
G
Describe the difference between nuclear and
H mitochondrial DNA
• Understand the use of DNA computerized databases in
criminal investigation
1
8 preservation of
• List the necessary procedures for the proper
bloodstained evidence for laboratory DNA
7 analysis
ISBN 1-256-36593-9
1
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KEY TERMS
amelogenin gene
amino acids
buccal cells
chromosome
complementary base
pairing
deoxyribonucleic
acid (DNA)
electrophoresis
epithelial cells
human genome
hybridization
low copy number
mitochondria
multiplexing
nucleotide
picogram
polymer
polymerase chain
reaction (PCR)
primer
proteins
replication
restriction fragment
length polymorphisms (RFLPs)
sequencing
short tandem
repeat (STR)
substrate control
tandem repeat
touch DNA
Y-STRs
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
266
CHAPTER 11
DNA
Abbreviation for deoxyribonucleic
acid—the molecules carrying the
body’s genetic information; DNA is
double stranded in the shape of a
double helix
chromosome
A rodlike structure in the cell
nucleus, along which the genes are
located; it is composed of DNA
surrounded by other material,
mainly proteins
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 G
original Jeffreys technique, the terms DNA profiling and DNA
typing came to be applied to describe
O this relatively new technology.
This discovery caught the imagination of the forensic science community because forensic
scientists have long desired to linkRwith certainty biological evidence such as blood, semen, hair,
or tissue to a single individual. Although
conventional testing procedures had gone a long way
D
toward narrowing the source of biological materials, individualization remained an elusive goal.
O
Now DNA typing has allowed forensic scientists to accomplish this goal. The technique is still
relatively new, but in the few yearsNsince its introduction, DNA typing has become routine in public crime laboratories and 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.
J
What Is DNA? E
S
Inside each of 60 trillion cells in the human body are strands of genetic material called chromosomes.
Arranged along the chromosomes,S
like beads on a thread, are nearly 25,000 genes. The gene is the
fundamental unit of heredity. It instructs
the body cells to make proteins that determine everyI
thing from hair color to our susceptibility to diseases. Each gene is actually composed of DNA
specifically designed to carry out aC
single body function.
Interestingly, although DNA A
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. But the major breakthrough
in comprehending how DNA works did not occur
L
until the early 1950s, when two researchers, James Watson and Francis Crick, deduced the strucE
ture of DNA. It turns out that DNA is an extraordinary molecule skillfully designed to carry out
the task of controlling the geneticItraits of all living cells, plant and animal.
Structure of DNA
polymer
A substance composed of a large
number of atoms; these atoms are
usually arranged in repeating units,
or monomers
nucleotide
The unit of DNA consisting of one
of four bases—adenine, guanine,
cytosine, or thymine—attached to
a phosphate–sugar group
G
H
Before examining the implications of Watson and Crick’s discovery, let’s see how DNA is constructed. DNA is a polymer. As we will learn in Chapter 12, a polymer is a very large molecule
made by linking a series of repeating
1 units.
In the case of DNA,
8 the repeating units are known as nucleotides. A nucleotide
is composed of a sugar molecule, a phosphorus-containing group, and a nitrogen-containing molecule called a base. Figure 11–1 7
shows how nucleotides can be strung together to form a DNA
strand. In this figure, S designates1the sugar component, which is joined with a phosphate group
to form the backbone of the DNA strand. Projecting from the backbone are the bases.
B
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 discusU
sion of DNA, we will designate each of these bases by the first letter of their names. Hence, A will
stand for adenine, C will stand for cytosine, G will stand for guanine, and T will represent thymine.
Again, notice in Figure 11–1 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; in fact, a DNA strand can be composed of a long chain with millions of
bases. The information just discussed was well known to Watson and Crick by the time they set
NUCLEOTIDES
ISBN 1-256-36593-9
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
DNA: THE INDISPENSABLE FORENSIC SCIENCE TOOL
about detailing the structure of DNA. Their efforts led to the discovery that the DNA molecule is
actually composed of two DNA strands coiled into a double helix. This can be thought of as
resembling two wires twisted around each other.
As these researchers 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 11–2).
COMPLEMENTARY BASE PAIRING The only arrangement possible in the double-helix configuration was the pairing of bases A to T and G to C, a concept that has become known as
complementary base pairing. Although A–T and G–C pairs are
G always required, there are no
restrictions on how the bases are to be 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 O
sequences are joined with their
complements in a double-helix configuration, they pair as follows:
R
T A T T
| | | |
A T A A
GD
T C A
| | | |
O
C A G T
G T A A
| | | |
C A T T
J
E
S
S
I
C
A
L
E
I
G
H
G
A
C
G
P
S
T
A
P
ISBN 1-256-36593-9
S
G
P
G
S
P
T
S
P
C
S
C
P
FIGURE 11–1
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.
complementary base pairing
The specific pairing of base A
with T and base C with G in
double-stranded DNA
C
T
P
S
S
N
Any base can follow another on a DNA strand, which means that the possible number of dif,
ferent 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
S
A
267
S
1
8
7
1
B
U
P
S
P
S
P
S
FIGURE 11–2
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.
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
268
CHAPTER 11
about 3 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.
WEBEXTRA 11.1
What Is DNA?
http://www.mycrimekit.com
DNA at Work
proteins
Polymers of amino acids that play
basic roles in the structures and
functions of living things
amino acids
The building blocks of proteins;
there are twenty common amino
acids; amino acids are linked to
form a protein; the types of amino
acids and the order in which they’re
linked determine the character of
each protein
The inheritable traits that are controlled by DNA arise out of its ability to direct the production of
complex molecules called proteins. Proteins are actually made by linking a combination of amino
acids. Although thousands of proteins exist, they can all be derived from a combination of up to 20
known amino acids. The sequenceG
of amino acids in a protein chain determines the shape and function of the protein. Let’s look at one
O example: The protein hemoglobin is found in our red blood
cells. It carries oxygen to our body cells and removes carbon dioxide from these cells. One of the
Rhemoglobin is shown in Figure 11–3(a). Studies of individuals
four amino acid chains of “normal”
with sickle-cell anemia show that D
this inheritable disorder arises from the presence of “abnormal”
hemoglobin in their red blood cells. An amino acid chain for “abnormal” hemoglobin is shown in
O
Figure 11–3(b). Note that the sole difference between “normal” and “abnormal” or sickle-cell
hemoglobin arises from the substitution
N of one amino acid for another in the protein chain.
The genetic information that determines the amino acid sequence for every protein manu,
factured in the human body is stored in DNA in a genetic code that relies on the sequence of bases
along the DNA strand. The alphabet of DNA is simple—A, T, G, and C—but the key to deciphering the genetic code is to know
J that each amino acid is coded by a sequence of three bases.
Thus, the amino acid alanine is coded by the combination C–G–T; the amino acid aspartate is
coded by the combination C–T–A;Eand the amino acid phenylalanine is coded by the combination
A–A–A. With this code in hand, we
S can now see how the amino acid sequence in a protein chain
is determined by the structure of DNA. Consider the DNA segment
S
I
The triplet code contained within this segment translates into
C
[C–G–T ] – [C–T–A] – [A–A–T] – [C–G–T]
alanine A aspartate phenylalanine alanine
–C–G–T–C–T–A–A–A–A–C–G–T–
Normal
hemoglobin
Sickle-cell
hemoglobin
1
valine
valine
2
histidine
histidine
3
leucine
leucine
4
threonine
threonine
5
proline
proline
6
glutamate
valine
7
glutamate
glutamate
(a)
(b)
L
phenylalanine — alanine
E
I
Interestingly, this code is not restricted
to humans. Almost all living cells studied to date use the
same genetic code as the languageGof protein synthesis.1
If we look at the difference between “normal” and sickle-cell hemoglobin (see Figure 11–3), we
H
see that the latter is formed by substituting
one amino acid (valine) for another (glutamate). Within
alanine — aspartate —
the DNA segment that codes for the production of normal hemoglobin, the letter sequence is
–[C–C–T]–[G–A–G]–[G–A–G]–
1
proline glutamate glutamate
8
7
–[C–C–T ]–[G–T–G]–[G–A–G]–
1proline valine glutamate
Bletter change (T has been substituted for A in valine) is the
Thus, we see that a single base or
underlying cause of sickle-cell anemia,
demonstrating the delicate chemical balance between
U
Individuals with sickle-cell disease carry the sequence
health and disease in the human body.
As scientists unravel the base sequences of DNA, they obtain a greater appreciation for the
roles that proteins play in the chemistry of life. Already the genes responsible for hemophilia,
1
Instructions for assembling proteins are actually carried from DNA to another region of the cell by ribonucleic acid
(RNA). RNA is directly involved in the assembly of the protein using the genetic code it received from DNA.
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
ISBN 1-256-36593-9
FIGURE 11–3
(a) A string of amino acids
composes one of the protein
chains of hemoglobin.
(b) Substitution of just one
amino acid for another in the
protein chain results in
sickle-cell hemoglobin.
or the protein chain
DNA: THE INDISPENSABLE FORENSIC SCIENCE TOOL
Duchenne muscular dystrophy, and Huntington’s disease have been located. Once scientists have
isolated a disease-causing gene, they can determine the protein that the gene has directed the cell
to manufacture. By studying these proteins—or the absence of them—scientists will be able to
devise a treatment for genetic disorders.
A 13-year project to determine the order of bases on all 23 pairs of human chromosomes (also
called the human genome) is now complete. Knowing where on a specific chromosome DNA
codes for the production of a particular protein is useful for diagnosing and treating genetic
diseases. This information is crucial for understanding the underlying causes of cancer. Also,
comparing the human genome with that of other organisms will help us understand the role and
implications of evolution.
G
O
Polymerase Chain Reaction (PCR)
R duplicated itself before cell
Once the double-helix structure of DNA was discovered, how DNA
division became apparent. The concept of base pairing in DNAD
suggests the analogy of positive
and negative photographic film. Each strand of DNA in the double helix has the same informaOfrom a positive.
tion; one can make a positive print from a negative or a negative
N
DNA Replication
,
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
J
by the principle of base pairing (A with T and G with C). The result is the emergence of two idenE 11–4). A cell can now pass on
tical copies of DNA where before there was only one (see Figure
its genetic identity when it divides.
S
Many enzymes and proteins are involved in unwinding the DNA strands, keeping the two
DNA strands apart, and assembling the new DNA strands. ForS
example, DNA polymerases are
enzymes that assemble a new DNA strand in the proper base sequence
determined by the origiI
nal or parent DNA strand. DNA polymerases also “proofread” the growing DNA double helices
C
for mismatched base pairs, which are replaced with correct bases.
Until recently, the phenomenon of DNA replication appeared
to be only of academic
A
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 relatively new laboratory technique
is known as polymerase
L
chain reaction (PCR). Put simply, PCR is a technique designed to copy or multiply DNA
E
strands.
269
human genome
The total DNA content found
within the nucleus of a human cell;
it is composed of approximately
three billion base pairs of genetic
information
replication
The synthesis of new DNA from
existing DNA
polymerase chain reaction
(PCR)
A technique for replicating or
copying a portion of a DNA strand
outside a living cell; this technique
leads to millions of copies of the
DNA strand
I
G
In PCR, small quantities of DNA or broken pieces of DNA found in crime-scene evidence can be
H can be accomplished in an
copied with the aid of a DNA polymerase. The copying process
ISBN 1-256-36593-9
The PCR Process
automated fashion using a DNA Thermal Cycler (see Figure 11–5). Each cycle of the PCR
technique results in a doubling of the DNA, as shown in Figure 11–4. Within a few hours,
30 cycles can multiply DNA a billionfold. Once DNA copies are1in hand, they can be analyzed by any of the methods of modern molecular biology. The
8 ability to multiply
small bits of DNA opens new and exciting avenues for forensic scientists to explore.
7 DNA recovered
It means that sample size is no longer a limitation in characterizing
from crime-scene evidence.
1
PCR is the outgrowth of knowledge gained from an understanding of how
B
DNA strands naturally replicate within a cell. The most important feature of PCR
is knowing that an enzyme called DNA polymerase can be directed
U 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:
–G–T–C–T–C–A–G–C–T–T–C–C–A–G–
–C–A–G–A–G–T–C–G–A–A–G–G–T–C–
Parent DNA
unravels
New double
helices formed
FIGURE 11–4
Replication of DNA. The
strands of the original DNA
molecule are separated,
and two new strands are
assembled.
#RIMINALISTICS !N)NTRODUCTIONTO&ORENSIC3CIENCE, Tenth Edition, by Richard Saferstein. Published by Prentice Hall. Copyright © 2011 by Pearson Education, Inc.
270
CHAPTER 11
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 the PCR process is to heat the DNA strands to about 94°C. At this
temperature, the double-stranded DNA molecules separate completely:
–G–T–C–T–C–A–G–C–T–T–C–C–A–G–
–C–A–G–A–G–T–C–G–A–A–G–G–T–C–
The second step is to add the primers to the separated strands and allow the primers to comG by lowering the test-tube temperature to about 60°C.
bine or hybridize with the strands
O
–G–T–C–T–C–A–G–C–T–T–C–C–A–G–
R
C–A–G–A
FIGURE 11–5
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 millionfold.
Courtesy of Life Technologies,
Carlsbad, CA. Reprinted with
permission.
primer
A short strand of DNA used to
target a region of DNA for
replication by PCR
hybridization
The process of joining two
complementary strands of DNA to
form a double-stranded molecule
WEBEXTRA 11.2
Polymerase Chain Reaction
D
C–C–A–G
O
–C–A–G–A–G–T–C–G–A–A–G–G–T–C–
N
The third step is to add the DNA polymerase and a mixture of free nucleotides (G, A, T, C) 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
J
segments.
E
–G–T–C–T–C–A–G–C–T–T–C–C–A–G–
S
C–A–G–A–G–T–C–G–A–A–G–G–T–C–
S
I
–C–A–G–A–G–T–C–G–A–A–G–G–T–C–
C
This completes the first cycle of the PCR technique, and the outcome is a doubling of the
A
number of DNA strands—that is, from one to two. The cycle of heating, cooling, and strand re–G–T–C–T–C–A–G–C–T–T–C–C–A–G
building is then repeated, resulting again in a doubling of the DNA strands. On completion of the
second cycle, four double-stranded DNA molecules will have been created from the orig…
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