LAB 11:
Human Genetics II:
Enzymatic Tests in Forensic Biology
I. Objectives:
This lab will introduce students to three of the most common enzyme-based assays that
forensic biologists use to analyze crime scene evidence for the presence of biological
fluids. By the end of today's lab, students should:

Be able to describe what enzymes are and what they do in the cell

Understand why different bodily fluids contain different types of enzymes

Be able to distinguish between a "presumptive test" and a "confirmatory
test" in forensic biology

Know how to examine and document biological crime scene evidence

Be able to perform presumptive enzymatic tests for blood, semen, and saliva

Be able to draw out the chemical reactions that underlie the presumptive
tests for blood, semen, and saliva and describe the role that enzymes play in
each assay

Know how to prepare an sexual assault evidence swab for microscopic
examination
II. Safety Considerations

The bodily fluids used in today's lab have not been screened for infectious biological
agents such as viruses and bacteria. THEREFORE, IT IS IMPORTANT TO WEAR
GLOVES AT ALL TIMES WHEN HANDLING THE EVIDENCE.

Discard all used evidence as biohazardous waste, according to the instructions in the
"Things to Do" section.
III. Introduction*
Enzymes
Enzymes are extremely important biomolecules that catalyze the chemical reactions in cells.
Almost all enzymes are proteins (a few are made of RNA) with complex three-dimensional
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structures that enable them to recognize their substrates in a "lock and key" interaction that
makes their action highly specific.
Although the human genome codes for thousands of enzymes, not all of them are produced
(expressed) in every tissue of the body. Some enzymes are needed in only one or a few
different tissue types and their presence in an evidence stain can therefore provide forensic
scientists with information about the type of bodily fluid that comprises the stain. This type of
information can be extremely important in determing what happened during the crime and can
also help forensic analysts narrow their search for DNA to stains that are likely to contain it.
The Crime
In today's lab, you will be analyzing two pieces of evidence from a crime in which a woman
was raped and murdered and her baby was kidnapped. A vaginal swab was taken from the
victim prior to autopsy and you will be analyzing this swab for the presence of semen. You will
also be analyzing a onesie that was found in the suspect's home to see if it contains saliva or
blood stains belonging to the kidnapped child. In each case, the presence of biological material
will enable you to determine if it is worthwhile examining the evidence further for the presence
of DNA. The presence of the suspect's DNA on the vaginal swab could help convict him of the
rape/murder, while the presence of the kidnapped baby's DNA on the onesie could help
convict him of the kidnapping (and help law enforcement locate the baby).
Presumptive versus Confirmatory Tests in Forensic Biology
At a crime scene and in a crime laboratory, it is often useful to perform a simple, highly
sensitive, but somewhat non-specific, test for a biological fluid. Such tests are called
presumptive tests and they help narrow the focus of the investigation to areas of the
evidence material that are likely to prove positive in later, confirmatory tests. In some cases
(e.g. tests on human urine) there is no confirmatory test and the presumptive test is the only
test available. Most of these tests are enzyme based, and rely on the fact that different bodily
fluids contain different types of enzymes, and the presence of these enzymes can be detected
by one or more fairly simple, inexpensive, and straightforward assays. Many of the assays are
simple, in fact, that they can be performed right at the crime scene – i.e. no laboratory is
required.
Confirmatory tests are usually more involved than presumptive tests and can rarely be
performed outside of a laboratory. However, confirmatory tests indicate that the substance
being screened for is actually present, whereas presumptive tests only indicate that the
substance may be present.
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Acid Phosphatase Presumptive Test for Semen
The most common presumptive test for the presence of seminal fluid is the AP (acid
phosphatase) or brentamine, test. Semen is particularly rich in acid phosphatase but the
enzyme is also present in vaginal secretions (esp. during pregnancy or if the woman has a
bacterial infection), feces, and some plants and fungi. However, these other substances tend
to produce a much weaker AP reaction than does semen.
It should also be noted that a negative AP test does not necessarily indicate that semen (and
sperm) are not present. If the evidence sample is from a vaginal swab, it may be AP negative
even though there are droves of sperm present. This is because AP, as well as p30 and PSA,
two other proteins used for semen identification, break down quickly in the vaginal cavity. By
contrast, sperm may last up to 5-7 days in the vagina after a sexual assault. Therefore, sperm
may be present long after AP and p30 are no longer detectable. A negative AP reaction should
thus never be used as an indication that semen is absent.
The brentamine test reagent (in our lab, the SERI AP spot reagent) is made by dissolving
equal amounts (in weight) of α-naphthyl acid phosphate monosodium salt and o-dianisidine
(tetrazotized) in brentamine buffer to a concentration of 2 g/L (0.2% solution). During the
reaction, naphthol is liberated from the α-naphthyl salt by acid phosphatase (if present) and
the naphthol couples with brentamine to form a purple azo dye. Fresh semen stains give a
fast, deep purple, color reaction.
α-naphthyl acid phosphate monosodium salt
sodium phosphate + naphthol
Acid phosphatase
napthol + Brentamine
Purple azo dye
Coupling reaction
Kastle-Meyer Presumptive Test for Blood
The Kastle-Meyer test is a quick presumptive test for the presence of blood. It is performed by
swabbing the suspected drop or stain with a cotton-tipped swab and then testing the swab to
see if the substance on it can reduce hydrogen peroxide. The heme group in the hemoglobin
of red blood cells reduces hydrogen peroxide, releasing oxygen free radicals in the process. In
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the assay, when blood is present, the released oxygen group oxidizes phenolphthalin (which is
colorless in its reduced form) to phenolphthalein (which is pink).
In the reaction, the swab is first moistened with distilled water and rubbed over a suspected
blood stain. A drop of Kastle-Meyer solution (containing phenolphthalin and sodium hydroxide)
is then placed on the swab. A drop of hydrogen peroxide is then added. If blood is present, the
hydrogen peroxide will be oxidized, hydrogen-hungry oxygen free radicals will be produced,
and the swab will turn pink as phenolphthalein is oxidized to phenolphthalein.
The rapid production of a pink color is an indication that blood may be present. However, a
negative reaction does not prove that blood is absent, particularly if the stain is old. The test is
presumptive because chemical oxidants (e.g. bleach) or any biological substance with
peroxidase or peroxidase-like activity will also result in a positive Kastle-Meyer assay.
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Amylase Presumptive Test for Saliva
Amylases are enzymes that break down starches into simple sugars. These sugars can then
be metabolized by a living organism as a carbon and energy source.
Starch is an insoluble compound comprised of long chains of branched (amylopectin) or
unbranched (amylose) glucose monomers. In amylose, the glucose monomers are connected
in a head-to-tail fashion, forming α-1,4 glycosidic bonds. (The related molecule, cellulose, has
β-1,4 bonds). In amylopectin, branching occurs, with an α-1,6 linkage every 24-30 glucose
monomer units. Around 2,500 glucose monomers are contained in a single starch molecule.
Starches have the molecular formula (C6H10O5)n, where "n" denotes the total number of
glucose monomer units.
The human amylases are coded by a gene family containing 5 tandemly-arranged genes on
chromosome 1. These genes are expressed in different tissues, primarily in oral saliva and by
the pancreas. The oral and pancreatic isoforms of amylase have slightly different amino acid
sequences and can be distinguished by isoelectric focusing and their specificity for monoclonal
antibodies.
Salivary amylase is known as ptyalin. It is found in humans and some other mammals but
not in dogs, or cats (since these common household pets are obligate carnivores). Ptyalin
starts the process of starch digestion by breaking down insoluble starches into soluble dextrins
and, ultimately, maltose (glucose dimers) as the food is being chewed. Salivary amylase is
denatured in the strong gastric acids of the stomach, but pancreatic amylase (amylopsin)
takes over in the small intestine to complete the breakdown process.
The amylase paper overlay assay for saliva tests to see if an evidence sample contains a
substance that can hydrolyze starch. A piece of filter paper is saturated with starch solution
and then laid on top of a portion of the evidence stain . If amylase is present in the stain,
some of it will transfer to the filter paper and hydrolyze the starch in the paper. This process is
visualized by spraying the filter paper with a solution of iodine. If no amylase is present, the
entire filter paper should stain blue-black. However, if amylase is present, areas of the filter
paper will no longer contain starch (due to the breakdown of the starch by the amylase
activity) and will remain white.
The current theory is that iodine stains the amylose component of starch blue-black because
iodine anions (I3- and I5-) slip inside the coiled structure of amylose. When they do this, they
transfer their charge to the amylose, and the energy level spacings in the resulting complex
absorb light in the visible portion of the spectrum.
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Amylose
http://www.lsbu.ac.uk/water/hysta.html
Amylase is an enzyme found at high concentrations in saliva. However, it is also found at
varying levels in most bodily tissues and fluids, including semen and vaginal fluid, and sweat.
Therefore, a positive amylase test (unfortunately) does not prove that saliva is
present. Therefore, the amylase overlay test is a presumptive test for saliva.
*NOTE: The information in the Introduction section of this lab was excerpted from: Forensic
Biology for the 21st Century: A Laboratory Manual for Serology and DNA, by Ruth Ballard and
Theresa Spear, copyrighted 2007. (Currently in publication review.)
IV. Things to Do
PART A. EXAMINATION OF EVIDENCE ITEM 1
1.
Put on a pair of gloves. If you have long hair, tie it back. Then get a bench diaper from
the front or side benches and place it down on your bench top to provide a clean
working surface.
2.
You have been provided with two evidence bags. The first bag (marked "Item 1") is an
infant's onesie, which you will be testing for the presence of saliva and blood. The
second bag (marked "Item 2") is a vaginal swab from a rape/homicide victim, which
you will analyzing for the presence of semen.
3.
Sign the evidence into your custody by writing your initials on the "To" line of the
Chain of Custody form on the top of each evidence bag. Then examine each bag. Are
the seals intact? Does the bag look like it is in good condition, or has it been
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damaged? Record your observations in your lab notebook and check the "sealed" box
on the Chain of Custody form on the top of the bag, using your Sharpie.
4.
Spray down your scissors with bleach solution and then wipe them down thoroughly
with a kim wipe. Then dip them in the distilled water and then in the beaker of
ethanol. Let them sit in the ethanol solution for about 10 seconds and then remove
them and wipe them down thoroughly with a kim wipe. You can now use them to cut
along the bottom of the bag containing Item 1.
5.
Carefully lay out Item 1 and examine it visually. Do you see any obvious stains? If so,
describe them in your lab notebook. Draw a diagram of the onesie in your notebook
and indicate the number and location of any visible stains. What color are the stains?
About how big are they?
6.
Also examine the onesie for any identifying information. Does the onesie have a tag?
If so, list the information on the tag(s), which may contain the size and brand of the
item. What color is the onesie? Does it have any identifying patterns on it? Are the
snaps open or shut? Does the onesie look new or old? Does it have any tears or other
damage? Record all this information in your lab notebook. Be sure to check both sides
of the onesie (front and back) as well as both surfaces (inside and out).
Why do you think criminalists record so much detail during their examination of crime
scene evidence?
PART B. KASTLE-MEYER TESTS ON ITEM 1 STAINS
1.
If any of the stains on the onesie appear to be rusty or red in color, they may be blood
stains and you should test them for the presence of blood using a Kastle-Meyer (K-M)
assay.
2.
To perform a K-M assay, moisten a cotton swab with distilled water and gently rub it
over the first reddish stain. Then add a drop of Kastle-Meyer Working Solution to the
tip of the swab. (Try to prevent getting the K-M solution on the wooden stick as it has
been bleached and will give a false positive reaction.)
3.
Add a drop of 3% hydrogen peroxide to the tip of swab. The quick formation of a pink
color change indicates the probable presence of blood.
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4.
Repeat steps 2-3 for the remainder of the stains, recording your observations after
each test.
5.
To make sure that your K-M reagents are working, you should also test a positive
control blood stain. Repeat the K-M test, wiping the swab against the positive control
blood filter provided to you by your instructor. Record your results in your notebook.
6.
For a negative control, use a cotton swab that has not been wiped on any stain or
substance. Repeat the K-M test on this swab and record your results.
7.
Were any of the reddish stains on the onesie positive for blood? If so, which ones?
Carefully record your results in your lab notebook, referring to the stains on the
drawing of the onesie you made during your evidence examination.
8.
When you are done, discard all your used swabs as biohazardous waste.
PART C. AMYLASE OVERLAY ASSAY ON FRONT OF ITEM 1
1.
Change your gloves and bench diaper.
2.
Saliva stains are very hard to see, so you will perform an amylase overlay assay on
Item 1 to try to find them if they are present. For this assay, you will need four large
Whatman filter paper circles - two to place on the front upper part of the onesie
(where saliva drool from a baby is most likely to be present), one for a positive
control, and one for a negative control. Label them L, R, +, and - using your Sharpie.
3.
Saturate all of the filter paper circles with starch solution (using the spray bottle of
starch provided).
4.
Clean your tweezers by spraying them with bleach solution and then dry them
thoroughly with a kim wipe. Then dip them in the beaker of distilled water and then in
the beaker of ethanol, letting them sit in the ethanol for about 10 seconds. Then wipe
them down again thoroughly with a kim wipe.
5.
Use the clean tweezers to lay the filter down side-by-side on the front side of the
onesie. Place the filter labeled "R" on the right side of the front of the onesie (from the
perspective of looking down on the onesie from above) and place the filter labeled "L"
on the left side of the front of the onesie, as shown at the top of the next page.
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6.
Prepare a positive control by gently swabbing the inside of your mouth with a cotton
swab and then wiping the cotton swab across the "+" filter paper. The filter paper can
be incubated right-side-up on the bench diaper.
7.
Your negative control will be a filter paper sprayed with starch but otherwise
untreated. It can also be incubated on the bench diaper.
8.
Allow the filter paper on the onesie to remain in contact with the onesie for about 20
minutes on your bench top.
9.
After the incubation period is complete, place all three filter papers in a moisture
chamber and incubate them at 37 degrees C for 1 hour. While you are waiting for this
incubation to finish, continue on to Part D.
10.
Remove the filter papers from the moisture chamber and dry for 10 minutes in a 37
degree oven.
11.
Spray the filter papers with Working Iodine Solution (provided in a spray bottle) to see
if a blue color develops. White areas on the filter paper are a positive indication
of the presence of amylase. The absence of white areas (the entire filer disk
is blue-black) is a negative indication of amylase.
12.
Record your results in your lab notebook. Did you detect any amylase? If so, on which
filter? Did you positive and negative controls give you the results you expected?
PART D. EXAMINATION OF EVIDENCE ITEM 2 AND ACID PHOSPHATASE ASSAY
1.
Change gloves and lay down a fresh bench diaper.
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2.
Spray down your scissors with bleach solution, wipe them thoroughly with a kim wipe,
dip them in the distilled water, and then dip them in the beaker of ethanol. Let them
sit in the ethanol solution for about 10 seconds and then remove them and wipe them
down thoroughly with a kim wipe. You can now use them to cut along the bottom of
the bag containing Item 2.
3.
Examine evidence item 2 and record your observations in your lab notebook.
4.
To test the swab for the presence of semen, wet down a Whatman filter paper circle
with distilled water (using the spray bottle provided). Then gently rub the swab
against the piece of filter paper, rolling it side-to-side. When you are done, DO NOT
DISCARD THE SWAB. Get a microcentrifuge tube of PBS (front bench) and carefully
remove the tip of the swab by breaking the wood stick just below the area where the
swab begins. Let the swab fall into the buffer in the tube and then cap the tube and
label it with your initials. You will need this tube for Part E, so set it aside for now.
5.
Prepare positive and negative control filter papers. For the positive control, use the
semen-stained positive control swabs provided. For the negative control, use a piece
of filter paper that has been saturated with water but has otherwise been untreated.
6.
Using a pipette bulb, saturate the filter papers with AP spot solution. The quick
appearance of a purple color change on the filter indicates the presence of semen.
7.
Record your results in your lab notebook.
8.
Discard all used filter papers as biohazardous waste.
PART E. PREPARATION OF ITEM 2 SWAB FOR MICROSCOPIC EXAMINATION FOR
SPERM
1.
A confirmatory test for semen can be performed by incubating an evidence stain in
PBS buffer at 4 degrees C with vigorous shaking for about 2 hours. This removes any
sperm that might be present on the swab or fabric and releases them into the buffer.
The swab or fabric swatch is then removed and the tube is spun in a microcentrifuge
to pellet the sperm. The sperm can then be stained and visualized microscopically (Lab
12).
2.
Bring your tube containing the swab and buffer to the front of the room. Your
instructor or TA will incubate the tubes (4 degrees C, shaking, 2 hours) and then spin
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the tubes for you. Next time, when you come to lab, your instructor will provide you
with the pellet from your swab extract and you will examine the pellet for
spermatozoa.
V. Lab Clean-Up

All leftover evidence (including the onesie) should be discarded as biohazardous
waste.

Used swabs and filters should be discarded as biohazardous waste

Gloves can be discarded in the trash unless they have come into direct contact with a
biological stain (in which case they should be discarded as biohazardous waste).
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