Difference between revisions of "Biomedical Forensics"

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= Objectives =
= Objective =
The objectives of this experiment are to extract DNA from a fruit sample, learn about fingerprinting, identify foreign substances, and perform blood typing to investigate a crime scene. The DNA will be extracted using the basic biochemical techniques for isolating, purifying, and digesting DNA molecules. The drug test will be completed using various testers. A blood typing kit will be used for the blood tests.
The objective of this experiment is to use the biomedical forensic techniques of extracting DNA, fingerprinting, identifying foreign substances, and blood typing to investigate a crime scene.


= Overview =
= Overview =
== Cellular Biology and Location of DNA ==
Biomedical forensic methods are used in criminal or civil cases. They are also used within the medical field to diagnose and treat diseases and injuries. Within criminal law, these techniques are used to identify suspects in criminal cases and to exclude individuals as suspects. DNA testing, in particular, is increasingly used to prove the innocence of people who have been wrongfully convicted of a crime.
DNA is the blueprint of life and is found in almost all living organisms. These organisms can be as simple as a single-celled bacteria or as complex as a multi-celled human; the human body contains approximately 50 trillion cells. There are two different types of cells: prokaryotes and eukaryotes. An example of prokaryotic organism is bacteria. Prokaryotic cells do not contain a nuclear membrane and so do not have a distinct nucleus. Only eukaryotic cells, which make up plants and animals, will be considered in this lab. Eukaryotic cells have a distinct, membrane-bound nucleus that isolates the DNA from the rest of the cell. The structure of plant cells is different from those of animal cells in structure and cellular contents. Only plant cells will be used in this experiment.


[[Image:DNA2.gif|frame|center|Figure 1: Cell membrane.]]
In this lab, DNA extraction will be introduced using basic biochemical techniques for isolating, purifying, and digesting DNA molecules. A toxicology report will be completed by observing chemical reactions using simulated reagents. A simulation kit will be used for the blood typing tests.


Plant cells are surrounded by a cell wall.  It has high mechanical strength and protects the cell.  Directly beneath the cell wall lies the plasma membrane (Figure 1), which contains the cytosol. The various cell organelles, including the nucleus, are found within the cytosol. The nucleus houses the DNA in the form of chromatin.
== DNA Extraction ==
<b>Deoxyribonucleic acid (DNA)</b> is found in almost all living organisms. In this lab, DNA will be extracted from plant cells in an introductory exercise to this lab.


[[Image:DNA3.gif|frame|center|Figure 2: Cell nucleus.]]
Plant cells are surrounded by structures called a cell wall and plasma membrane that protect the cell. Inside the cell sits various units called organelles; DNA is located in an organelle called the nucleus.


Chromatin is the active form of DNA in the cell when it is not preparing for cell division. It is comprised of DNA wrapped around protein particles called histones.
The <b>DNA extraction</b> process can be divided into three major steps: breaking open the cell wall, destroying membranes within the cell, and precipitating the DNA out of the solution.


[[Image:DNA1.gif|frame|center|Figure 3: Cell structure.]]
The first step in DNA extraction is to <b>break the cell wall (cell lysis)</b> to expose the DNA within the cell. Cell walls are rigid structures made of cellulose and thus require mechanical methods to break apart. In this experiment, DNA will be extracted from a strawberry that will be mashed manually.


== DNA Extraction Technique ==
The second step in DNA extraction is to <b>destroy the plasma membrane within the cell</b>. The cell's plasma membrane is made of phospholipid bilayers that are made of fat. To disrupt them, the mesh of fat molecules is broken up with a surfactant (soap). The structure of soap is similar to the structure of fat and grease and lowers the surface tension between two fluids when acting as a surfactant.
One of the many goals in this experiment is to extract the DNA from a fruit sample. Some knowledge of the scientific background behind DNA extraction is needed to do this.


The DNA extraction process is a fairly simple biochemical procedure that can be divided into three major steps: breaking open the cell (lysis), destroying membranes within the cell, and precipitating the DNA out of the solution.
A soap molecule has two parts: a head and a tail. The head is polar and is attracted to water (hydrophilic), while the tail is nonpolar and attracted to oil and fat (hydrophobic). When soap molecules are in water, they group themselves into micelles that have a roughly spherical structure in which all the polar heads point outwards (toward water) and all the nonpolar tails point inwards at the center of the sphere (away from the water) (Figure 5).  


The following sections describe how each step relates to the physical and biochemical properties of DNA.
[[Image:DNA4.gif|thumb|500px|center|Figure 5: Soap Micelle]]


== Cell Lysis (Breaking Open the Cell Wall and Membranes) ==
The soap molecules effectively trap the fat molecules inside the micelles and dissolve the cell membranes. The soap molecules orient themselves so that their head associates with the tail of the phospholipid bilayer. In this way, the soap breaks up the bilayer molecule by molecule (Figure 6).
Plant cells have a very rigid external structure &mdash; the cell wall &mdash; which protects it. To get to the DNA, the very first step would be to break open that wall.


The cell wall is the first barrier in that must be broken to extract the DNA molecule inside the cell. It is very rigid and acts as a protector and filter. It is made of cellulose, and is responsible for making wood hard and durable. To destroy the cell wall, a mechanical method is used to break apart the cellulose molecules. In this experiment, the fruit sample is mashed '''manually'''.
[[Image:DNA14.gif|thumb|500px|center|Figure 6: Phospholipid Bilayer]]


== Destroying Membranes Within the Cell ==
The last step in DNA extraction is <b>precipitating the DNA</b>. When the plasma membrane is successfully disrupted, the DNA is released from the cells into the solution along with protein molecules and other cellular miscellany. With the cell's contents mixed into a solution, the DNA can be separated from the rest of the contents. This process is called precipitation. Salt is used because it disrupts the structure of the proteins and carbohydrates found in the solution. Also, the salt provides a favorable environment to extract the DNA by contributing positively-charged sodium ions that neutralize the negative charge of DNA.
The cell's plasma membrane is made of phospholipid bilayers which are made up of fat. To disrupt them, that mesh of fat molecules is broken up with soap. The structure of soap is very similar to that of fat and grease.


[[Image:DNA4.gif|frame|right|Figure 4: Soap micelle.]]A soap molecule has two parts: a head and a tail. The head is polar and is attracted to water while the tail is non-polar and is attracted to oil and fat. When soap molecules are in water, they group themselves into micelles &mdash; a roughly spherical structure in which all the polar heads point outwards (in contact with water) and all the non-polar tails point inwards at the center of the sphere (away from the water). They can effectively trap the fat molecule inside the micelle and dissolve the cell membranes. How does this micelle break down the phospholipid bilayer? The molecules in the phospholipid bilayer (Figure 5) also contain molecules that are made up of a hydrophobic head and a hydrophilic tail. The soap molecules orient themselves so that their head associates with the tail of the phospholipid bilayer. In this way, the soap is able to break up the bilayer molecule by molecule.
After adding salt and soap, the extracted DNA cannot be seen as it is too small to distinguish from the rest of the solution. To make the DNA visible, alcohol is added since it cannot dissolve the DNA. A translucent white substance will begin to form at the top: this is the DNA (Figure 7). Once the precipitate is thick enough, it can be spooled out. This simple procedure is a rough extraction process that needs further steps before the DNA can be successfully run on a gel for analysis. This analysis will not be performed in this lab.  


[[Image:DNA14.gif|frame|center|Figure 5: Phospholipid bilayer.]]
[[Image:DNA5.gif|frame|500px|center|Figure 7: Extracted DNA]]
<br style="clear: both;" />


== Precipitating the DNA ==
== Fingerprints ==
When the membrane is successfully disrupted, the DNA is released from the cells into the solution along with protein molecules and other cellular miscellanea.


The DNA molecule is a double-helical polymer consisting of a sugar-phosphate backbone with nitrogenous bases running perpendicular to the backbone. These bases, often represented by letters &mdash; A (adenine), G (guanine), C (cytosine), and T (thymine) &mdash; are the elementary components making up the coded genetic information (Figure 7). The base sequence acts as the instruction manual of the cell, directing it on how to make proteins and other important molecules that an organism needs to survive and function.
<b>Fingerprints</b> are unique patterns consisting of friction ridges (raised) and furrows (recessed) that appear on the pads of the fingers and thumbs and are often used for identification purposes in forensics. Prints from palms, toes, and feet are also unique, but these are used less often for identification so this guide focuses on prints from the fingers and thumbs.


With the cell's contents mixed into a solution, the DNA is separated from the rest. This process is called precipitation. Salt is used because it disrupts the structure of the proteins and carbohydrates found in the solution. Also, the salt provides a favorable environment to extract the DNA by contributing positively charged sodium ions that neutralize the negative charge of DNA. After the addition of salt and soap, the manner by which the DNA is being extracted out of the solution cannot be seen as it is too small to distinguish from the rest of the solution. To aid in precipitating the DNA, alcohol is added since it cannot dissolve DNA. A translucent white substance will begin to form at the top: this is DNA. Once it is thick enough, it can be spooled out. This simple procedure is a rough extraction process that needs further purification before it can be successfully run on a gel for analysis.
The fingerprint pattern, such as the print left when an inked finger is pressed onto paper, is made from the friction ridges on that particular finger. Friction ridge patterns are grouped into three distinct types – loops, whorls, and arches – each with unique variations, depending on the shape and relationship of the ridges.


[[Image:DNA5.gif|frame|center|Figure 6: Extracted DNA.]]
<b>Loops</b> are prints that recurve back on themselves to form a loop shape (Figure 8). Divided into radial loops (pointing toward the radius bone, or thumb) and ulnar loops (pointing toward the ulna bone, or pinky), loops account for approximately 60% of pattern types.


[[Image:Loops.png|thumb|500px|center|Figure 8: Loop Fingerprint]]


== Restriction ==
<b>Whorls</b> form circular or spiral patterns, like tiny whirlpools (Figure 9). There are four groups of whorls: plain (concentric circles), central pocket loop (a loop with a whorl at the end), double loop (two loops that create an S-like pattern) and accidental loop (irregularly shaped). Whorls make up about 35% of pattern types.
Often times, larger fragments of DNA are cut, or restricted, to extract a particular fragment. This is made possible by the action of restriction enzymes, which are used by bacteria to cut up foreign or enemy DNA. Restriction enzymes are catalytic proteins that recognize specific palindromic DNA sequences and cut the double-stranded DNA at particular sites. The sites that the restriction enzymes recognize are called restriction sites. There are many different types of restriction enzymes. Each type recognizes a different restriction site. In this lab, Lambda DNA, which is a commercially available DNA normally found in a virus called Phage Lambda, will be restricted with the restriction enzyme BamH1.


[[Image:DNA9.gif|frame|center|Figure 7: Nitrogenous bases of DNA.]]
[[Image:Whorls.png|thumb|500px|center|Figure 9: Whorl Fingerprint]]


{| class="wikitable" style="text-align: center; margin-left: auto; margin-right: auto;"
<b>Arches</b> create a wave-like pattern and include plain arches and tented arches (Figure 10). Tented arches rise to a sharper point than plain arches. Arches make up about 5% of all pattern types.
|+style="caption-side: bottom"|Figure 8: Restriction enzymes.
!Enzyme!!Source!!Recognition Sequence!!colspan="2"|Cut
|-
|BamH1||Bacillus amyloliquefaciens
|<pre>5'GGATCC
3'CCTAGG</pre>
|align="left"|<pre>5'-----G
-------CCTAG</pre>
|align="right"|<pre>GATCC------
G-----5'</pre>
|-
|EcoRI||Escherichia coli
|<pre>5'GGATCC
3'CCTAGG</pre>
|align="left"|<pre>5'-----G
-------CCTAG</pre>
|align="right"|<pre>GATCC------
G-----5'</pre>
|-
|HindIII||Haemophilus influenzae
|<pre>5'AAGCTT
3'TTCGAA</pre>
|align="left"|<pre>5'---A
3'---TTCGA</pre>
|align="right"|<pre>AGCTT---3'
A---5'</pre>
|}


== Gel Electrophoresis ==
[[Image:arches.png|thumb|500px|center|Figure 10: Arch Fingerprint]]
[[Image:DNA7.gif|frame|right|Figure 9: Agarose gel.]]The technique of DNA electrophoresis (Figure 10), will be performed on uncut and cut Lambda DNA, a commercially available DNA on an agarose gel, to visualize the characteristic banding patterns that differentiate between different DNA fragments. Gel electrophoresis is a technique used for separating molecules based on their charge and molecular weight.  The sample is loaded in a gel matrix and an electric field is applied across it. The electric field enables the DNA, which is negatively charged to migrate to the end, which is positively charged. Opposites attract and so the negatively charged DNA is attracted to the positive end of the gel. Lighter molecules will migrate to the opposite end of the gel faster than heavier molecules.


[[Image:DNA6.gif|frame|center|Figure 10: Gel electrophoresis.]]<br style="clear: both;" />
== Blood Typing ==


The following is a gel after the samples have been run. Each column is referred to as a lane, representing one sample each. The individual bands (Figure 11) contain fragments of DNA that are identical in weight.
Even though all red blood cells are made of similar elements, not all blood cells are alike. There are eight different types of blood, each based on the presence or absence of three types of <b>antigens</b> (A, B, and Rh) (Figure 11). Generally, antigens induce an immune response to foreign substances in the body. The <b>A and B antigens</b> determine the type of blood while the <b>Rh antigen</b> determines if the blood is positive or negative with the Rhesus factor, which is associated with hemolytic diseases and incompatible blood transfusions.


[[Image:DNA8.gif|frame|center|Figure 11: DNA banding pattern following gel electrophoresis.]]
*The ABO Blood Group System
**Group A has only the A antigen on red cells
**Group B has only the B antigen on red cells
**Group AB has both A and B antigens on red cells
**Group O has neither A nor B antigens on red cells


== Fingerprints ==
Positive (+) blood types have the Rh factor and negative (-) blood types do not have the Rh factor.
Fingerprints are unique patterns, made by friction ridges (raised) and furrows (recessed), which appear on the pads of the fingers and thumbs. Prints from palms, toes and feet are also unique; however, these are used less often for identification, so this guide focuses on prints from the fingers and thumbs.


The fingerprint pattern, such as the print left when an inked finger is pressed onto paper, is that of the friction ridges on that particular finger. Friction ridge patterns are grouped into three distinct types—loops, whorls, and arches—each with unique variations, depending on the shape and relationship of the ridges:
[[Image:Lab_biomed_1.PNG|thumb|500px|frame|center|Figure 11: Different Blood Types]]


Loops are prints that recurve back on themselves to form a loop shape. Divided into radial loops (pointing toward the radius bone, or thumb) and ulnar loops (pointing toward the ulna bone, or pinky), loops account for approximately 60 percent of pattern types.
= Materials and Equipment =
 
[[Image:Loops.png|frame|center|Figure 12: A looped fingerprint.]]<br style="clear: both;" />


Whorls form circular or spiral patterns, like tiny whirlpools. There are four groups of whorls: plain (concentric circles), central pocket loop (a loop with a whorl at the end), double loop (two loops that create an S-like pattern) and accidental loop (irregular shaped). Whorls make up about 35 percent of pattern types.
*Strawberry
 
 
[[Image:Whorls.png|frame|center|Figure 13: A whorled fingerprint.]]<br style="clear: both;" />
 
 
Arches create a wave-like pattern and include plain arches and tented arches. Tented arches rise to a sharper point than plain arches. Arches make up about five percent of all pattern types.
[[Image:arches.png|frame|center|Figure 14: An arched fingerprint.]]<br style="clear: both;" />
 
= Materials and Equipment =
<div style="float: right;">
<div style="display: inline-block;">[[Image:DNA10.gif]]<br />[[Image:DNA11.gif]]</div>
<div style="display: inline-block;">&emsp;&emsp;&emsp;</div>
<div style="display: inline-block;">[[Image:DNA12.gif]]<br />[[Image:DNA13.gif]]</div>
</div>
*Materials and Equipment
*Fruit sample
*Non-iodized table salt (NaCl)
*Non-iodized table salt (NaCl)
*Hand soap (clear, unscented)
*Hand soap (clear, unscented)
Line 122: Line 74:
*Plastic cups
*Plastic cups
*Ziploc bag
*Ziploc bag
*Iron Magic Wand
*Iron magnetic wand
*Iron powder
*Iron powder
*Blood typing kit
*Blood typing kit
*White Paper
*Pipettes
*String
*Beaker
*Test tubes
*Microcentrifuge tubes
<!--*Plexiglass pieces-->
*Baby wipes
*Meter stick
*Meter stick


= Procedure =


<br style="clear: both;" />
Watch the following video: [https://drive.google.com/file/d/18f1tHzFJg5bnU5ky9-HWcFqvm9FePp_K/view?usp=sharing Biomedical Forensics Part 1]


= Procedure =
== Forensic Academy ==
== Forensic Academy ==
Before you can go out into the field and investigate your first crime, you must first go through basic training to become an official EG forensic scientist. In this time, three tests will be done to enhance your knowledge of the forensic science and how the modern forensic scientist would start an investigation.


=== Part 1: DNA Extraction ===
Basic training in three forensic techniques will be completed before the crime scene investigation is conducted.
<ol>
 
<li>Put a bottle of isopropyl alcohol in a freezer. We’ll come back to it later. Measure 6T (90 ml) of water into a small glass container.</li>
=== 1. DNA Extraction ===
<li>Stir in a ¼-tsp salt and mix until the salt dissolves. This is the extraction mixture.</li>
 
<li>Stir in a ¼-tsp salt and mix until the salt dissolves. This is the extraction mixture.</li>
# Place 200 ml of distilled water in a beaker.
<li>Place one banana into a plastic zipper-lock bag.</li>
# Add 2 tsp (10 ml) of dish soap to the water.
<li>Pour the extraction mixture into the bag with the banana.</li>
# Stir in ¼ tsp of salt and mix until the salt dissolves. This is the extraction mixture.
<li>Remove as much air from the bag as possible and seal it closed.</li>
# Place one strawberry into a Ziploc bag.
<li>Use your hands and fingers to mash, smash, and moosh the strawberry inside of the bag. You don’t want any large pieces remaining.</li>
# Pour the extraction mixture into the bag with the strawberry.
<li>Pour the resulting strawberry pulp and extraction mixture through a strainer and into a medium glass bowl or similar container.</li>
# Remove as much air from the bag as possible and seal it closed.
<li>Use a spoon to press the mashed bits of strawberry against the strainer forcing even more of the mixture into the container. 10. From the container it’s in now, pour the extraction mixture into a smaller glass container that holds ¼- to ½-cup (50-100 ml) of fluid. This will help to isolate the DNA on the surface of the mixture.</li>
# Mash the strawberry inside the bag manually. Crush any large pieces.
<li>Add 1 tsp (5 ml) of the chilled isopropyl alcohol to the solution and hold the mixture at eye level. You’re looking for a separation of material that shows up as a white layer on top. That’s the DNA of the strawberry!</li>
# Pour the resulting strawberry pulp and extraction mixture through a strainer and into a beaker.
<li>Use the tweezers to gently remove the DNA from the solution and lay it on a dish to examine.</li>
# Use a plastic spoon to press the mashed bits of strawberry against the strainer forcing as much of the mixture as possible into the container.
</ol>
# Pour the extraction mixture into a test tube. This will help isolate the DNA on the surface of the mixture.
# Add 1 tsp (5 ml) of the chilled isopropyl alcohol to the solution (request the isopropyl alcohol from a TA) and hold the mixture at eye level. Look for the separation of DNA that shows up as a white layer on the surface of the mixture.
# If the separation of DNA cannot immediately be seen, set the small container aside for a few minutes and examine it again later.
 
=== 2. Fingerprinting ===


=== Part 2: Fingerprinting ===
# Obtain a clean piece of Plexiglass and thoroughly clean it with a baby wipe.
<ol>
# Apply a thumb onto the glass and put pressure onto the glass while holding the glass by its edges. Keep applying pressure for a few seconds.
<li>Obtain a clean piece of Plexiglass and thoroughly clean it with a baby wipe.</li>
# Hold the glass against the light to view the imprint to determine if it is visible. If not, clean the glass and repeat the process.
<li>Apply your thumb onto the glass, and put pressure onto the glass, while holding the glass by its edges.</li>
# Take the iron filament and spread a generous amount on the region of the imprint.
<li>Keep applying pressure for 30 seconds.</li>
# Take the magnetic wand and slowly pull the top across the iron filament to extract the excess iron filament from the Plexiglass piece.
<li>Once the time is up, hold the glass against the light to view the imprint and is it if it came out clearly. If not, clean the glass and redo the process.</li>
# Place the excess iron filament back into the container and seal the container.
<li>Take the fingerprint powder and spread a generous amount in the region of the imprint.</li>
# Closely observe the remaining fingerprint and identify the fingerprint type.
<li>Take the magic wand and slowly lift the top to extract the excess iron filament from the glass panel.</li>
# Take a picture of the fingerprint on a white background.
<li>Place the excess back into the black powder container and seal the container.</li>
<li>Closely observe the remaining fingerprint and identify which type of fingerprint it is.</li>
</ol>


=== Part 3: Drug Testing ===
=== 3. Toxicology Report ===
<ol>
<li>Obtain lab samples of the suspect drug.</li>
<li>Separate the drug sample into 15 test tubes, label each test tube A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2, D3, E1, E2, E3.</li>
<li>Obtain the 5 drug identifiers from the TAs. Identifiers tests for the following drugs:
*Cannabis
*Heroin
*MDMA (Ecstasy)
*Cocaine
*Lysergic Acid Diethylamide (LSD)</li>


<li>Take all the A-labelled test tubes with the drug sample and place 5 droplets of the Cannabis.</li>
# Obtain samples of the suspect drug from a TA.
<li>Stir the test tube for 30 seconds.</li>
# Separate the drug sample into five microcentrifuge tubes and separately label the microcentrifuge tubes A, B, C, D, or E.
<li>Observe each test tube. If the test tube has formed a fizzing substance, the powder contains that drug.</li>
# Obtain the five drug reagents from a TA. The reagents test for the following drugs.
<li>Repeat Step 4-6 for each of the identifiers (Test Tube B-Heroin, Test Tube C-MDMA, Test Tube D- Cocaine, Test Tube E-LSD).</li>
#: <br>
#: Drug A: Cannabis
#: Drug B: Heroin
#: Drug C: MDMA (Ecstasy)
#: Drug D: Cocaine
#: Drug E: Lysergic Acid Diethylamide (LSD)
# Take the A-labelled microcentrifuge tube with the drug sample and place five droplets of the cannabis reagent.
# Observe the test tube. If a reaction is observed from adding the reagent, the tube contains that drug. The microcentrifuge tube might need a quick shake to catalyze the reaction.
# Repeat Steps 4 and 5 for each of the drug reagents.


</ol>


=== Part 4: Blood Splatter Analysis ===
<b>Congratulations, the Forensic Academy is completed and the graduates are certified to participate in the crime scene investigation!</b>
==== Distance Test ====
<ol>
<li>Measure a height of 15cm.</li>
<li>Using a pipette, administer a droplet of blood onto a piece of white paper. </li>
<li>Record the radius of the resulting droplet.</li>
<li>Repeat steps 1-3 for heights of 30cm, 60cm, 90cm, 135cm, 180cm.</li>
</ol>
==== Angle Test ====
<ol>
<li>From a height of 60 cm, administer a drop of blood at an angle of 90° from the horizon.</li>
<li>Record the length of the resulting droplet.</li>
<li>Repeat the step 1-2 for angles of 75°, 60°, 45°, 30°, 15°, 10° from the horizon.</li>
</ol>


== Crime Scene Investigation ==


<b>Congratulations, you have now completed the Forensic Academy and now are certified to fight crime in the greater EG area!</b>
It was a warm and stormy night on September 5, 1975. The EG1004 office was quiet as one TA was pulling a late night shift to prepare the classroom for the next morning’s lab. There was a loud crash in the background. The room went dark. The next morning, there was police tape barring entry to the entire room. Someone had murdered Ruhit Roy. At the crime scene, police discovered a pool of blood, along with the suspected murder weapon – a shard of glass – near the body. When the investigation yielded no results, Ruhit’s murder was declared a cold case. Watch the following video: [https://drive.google.com/file/d/15w4Wzz0CZ6b40Don5Qm-U--HXUYZ35i5/view?usp=sharing Biomedical Forensics Part 2]


== Crime Scene ==
After many years, Ruhit’s case is finally being reopened. Watch the following video: [https://drive.google.com/file/d/1QHC4Wt8yRMiDP3if-xIbV7D5mQergxF4/view?usp=sharing Biomedical Forensics Part 3]
It was a warm and stormy night on July 23rd, 2017. The EG office was quiet as one TA was pulling a late night shift to prepare the classroom for the next morning’s lab. There is a loud crash in the background. The room went dark. The next morning, there was police tape engulfing the entire room. Someone had murdered Roy, and now it is up to you to find his killer.


At the crime scene, police discovered a pool of blood, along with the suspected murder weapon &mdash; a hammer &mdash; near the body. They have narrowed down the suspects to the 26 people who had access to the office that late at night. Here is an [[Media:Biomedical Forensics Excel.xlsx|Excel Document]] of the basic information of the suspects.
<b>Download the [https://docs.google.com/spreadsheets/d/10Mmt_5Xxx_TjEqNTz_aRtXNUEmbcAcXj/edit?usp=sharing&ouid=100215762625371235485&rtpof=true&sd=true Suspect Information Sheet] to get started on the investigation.</b>


It is up to you to find the killer before this case gets cold, and the murderer is never found.
=== 1. Biological Dimensional Analysis ===
# Obtain a yard stick and measure the length of a foot with shoes on.
# Measure the length of a hand from the tip of the middle finger to the bottom of the palm.
# Repeat Steps 1 and 2 for the additional members in the group.
# Calculate and compare the ratios of the hand length to foot length and determine the average foot to hand length ratio based on the data obtained.
# Using the average foot length to hand length ratio, approximate the hand length of the murderer using the 11 in footprint found at the crime scene. Record the estimated measurements of the killer (Figure 12).


=== Part 1: Biological Dimensional Analysis ===
[[Image:Foot.png|thumb|600px|center|Figure 12: An 11" Footprint Left at the Crime Scene]]
<ol>
<li>Obtain a piece of string and make a knot that is equal to the length of your foot.</li>
<li>Compare this string to various part of your body (ie. size of your head, arm length, leg length, hand size, overall height, etc.)</li>
<li>Repeat steps for each member in group.</li>
<li>Compare the ratios of each body part ratios within the group.</li>
</ol>


=== 2. Toxicology Report ===


Using the average ratio of each person, approximate the dimensions of the murderer using the image below, taken at the scene of the crime.
# To collect important information on the murder, the victim’s blood will be tested  to determine the drug that killed him.
# Put a small sample of Roy’s blood into five microcentrifuge tubes. Use the drug reagents to determine which drug was injected into his bloodstream by the killer.
# Record the drug that was used on the victim.


[[Image:Footprint.jpg|500px|frame|center|Figure 1: Cell membrane.]]
<font color="red"><b>'''Note: Please use caution when using the microcentrifuge tubes, as they can explode.'''</b></font>


=== Part 2: Blood Typing ===
Watch the following video: [https://drive.google.com/file/d/1yCpCj2oNPDNIy-90OfRXMC4ZazPpiMRk/view?usp=sharing Biomedical Forensics Part 4]
<ol>
<li>Obtain three vials of each of the following: twenty-six blood samples of each suspect, blood sample found on the weapon and the crime scene, and the victim’s uncontaminated blood.</li>
<li>Gather the three indicator beakers labelled the Anti-A Serum, Anti-B Serum, Anti-RH serum.</li>
<li>Using a pipette, administer 5 drops of the Anti-A Serum into the vial labelled Suspect 1 A.</li>
<li>Shake the vial for 15 seconds.</li>
<li>If a precipitate forms (cloud material), then the blood type is Blood type A. If the liquid appears to be more diluted (more liquidity), then the vial is not Blood type A.</li>
<li>Repeat steps 3-5 for each person and for each of the indicators.</li>
<li>Using the blood found on the weapon, at the crime scene and the uncontaminated blood to determine the possible blood type of the murderer.</li>
</ol>


=== Part 3: Fingerprint Test ===
=== 3. Blood Typing ===  
<ol>
<li>Following a similar procedure as in training, carefully obtain the murder weapon. </li>
<li>Carefully apply the iron filament at the rim of the bat, using a piece of white paper to catch the excess filament.</li>
<li>Using the Magic wand, carefully remove the excess filament.</li>
<li>Fingerprint residue should be left behind after removing the excess filament; if not, redo steps 2 and 3.</li>
<li>Observe the fingerprint, noting what feature it contains and what type it is.</li>
</ol>


# Obtain the blood sample from the crime scene and from all 10 suspects and the victim along with their respective A, B, and Rh serum capsules.
# Using a pipette, administer three drops of Suspect 1’s blood into all three ovals of the test tray.
# Using a pipette, administer a one drop of the A serum into the oval labeled A, administer one drop of the B serum into the oval labeled B, and administer one drop of the Rh serum into the oval labeled Rh. Wash the pipette out with water after administering each serum and between each suspect to prevent cross-contamination.
# If the blood sample exhibits a reaction to the indicator (bubbles or becomes cloudy), then the blood type matches that indicator type. If the liquid appears to be more diluted (more watery or thin), then the blood type does not match that indicator type. For example, if a precipitate forms for A and Rh for a blood sample, then it is A+. If a precipitate forms for A and B, but not Rh, that blood type is AB- If the blood samples do not react to any serum, then that blood type is O-.
# If a precipitate forms for A and B, but not Rh, that person’s blood type is AB-.
# Repeat Steps 2-5 for each suspect. Do <b>not</b> mix up the serums as that will determine the wrong blood type.
# The blood found at the scene of the crime was A-. Test Roy’s blood to make sure that the blood at the scene is the killer’s blood and not Roy’s blood.
# Add the blood types of each person to the Suspect Information Sheet to determine if any suspect’s blood type matches the blood type found at the crime scene.


Using all the information gathered in these three experiments, filter through the provided excel to determine who the killer is.
=== 4. Fingerprint Test ===
 
# Carefully obtain the murder weapon (the shard of glass).
# Carefully apply the iron filament at the rim of the glass shard, trying not to waste filament.
# Using the magnetic wand, carefully remove the excess filament.
# Fingerprint residue should be left behind after removing the excess filament; if not, repeat Steps 2 and 3.
# Observe the fingerprint, noting its features and type. Determine which person it corresponds to in the Suspect Information Sheet.
 
Using all the information gathered in the analyses, use the data provided in the Suspect Information Sheet to determine who the killer is. Tell a TA who the alleged killer is.


= Assignment =
= Assignment =
== Individual Lab Report ==
== There is no lab report for Lab 11. ==
Follow the lab report guidelines laid out in the page called [[Specifications for Writing Your Lab Reports]] in the Technical Communication section of this manual. The following discussion points should be addressed in the appropriate section of the lab report:
 
* Discuss the structure of a plant cell.
<!--{{Labs:Lab Report}}
* Justify the use of salt, soap, and alcohol in the extraction procedure.
 
* Explain how to reach the DNA and the barriers that were overcome to get to it.
* Discuss the importance of biomedical forensics
* Describe the major techniques used in this lab: DNA Restriction, Gel Electrophoresis, etc.
* Explain the three steps of DNA extraction
* Important properties of DNA directly having an impact on the extraction procedure.
* Describe the most common types of fingerprints
* Clearly describe the procedural steps the way they were carried out in lab.
* Explain what causes different blood types
* Describe the steps carried out with the TA.
* Justify the use of salt, soap, and alcohol in the extraction procedure
* Explain the test results for the Biuret test and Benedict's test.
* Discuss how knowing the victim's blood type helped find the killer
* Include appropriate figures to support the observations made.
* Discuss the important properties of DNA directly having an impact on the extraction procedure
* Specify the location in the gel of the DNA sample that belonged to the team.
* Clearly describe the procedural steps that were carried out in lab
* Compare the results with the control group.
* Describe the steps carried out with the TA
* Include pictures of the extracted DNA, fingerprint on the murder weapon, and drug present in the victim’s blood
* Include the hand size and blood type of the killer
* How would procedure change if meat was used instead of fruit?
* How would procedure change if meat was used instead of fruit?
* Discuss improvements that could be made to the experiment
* Discuss what part of the lab each individual member completed for the group and how it was important to the overall experiment.


{{Lab notes}}
{{Labs:Lab Notes}}-->


== Team PowerPoint Presentation ==
== Team PowerPoint Presentation ==
Follow the presentation guidelines laid out in the page called [[EG1003 Lab Presentation Format]] in the Introduction to Technical Presentations section of this manual. When preparing the presentation, consider the following points:
 
* Rely heavily on graphics and pictures.
{{Labs:Team Presentation}}
* Make sure the Experimental Work is described simply and thoroughly.
 
* Discuss the real-life application of DNA sequencing.
* Rely heavily on graphics and pictures
* Demonstrate clear understanding of each procedural step carried out and why it worked.
* Make sure the experimental work is described simply and thoroughly
* Discuss the real-life application of DNA extraction
* Demonstrate clear understanding of each procedural step carried out and why it worked


= References =
= References =
<p>[http://www.vernier.com http://www.vernier.com]</p>
<p>[http://www.vernier.com http://www.vernier.com]</p>
<p>[http://www.invitrogen.com http://www.invitrogen.com]</p>
<p>[http://www.invitrogen.com http://www.invitrogen.com]</p>
<p>Nasco Website</p>
<p>[http://en.wikipedia.org http://en.wikipedia.org]</p>
<p>[http://en.wikipedia.org http://en.wikipedia.org]</p>
<p>The Science Creative Quarterly</p>
<p>[http://www.hhmi.princeton.edu/documents/labprotocols http://www.hhmi.princeton.edu/documents/labprotocols]</p>
<p>[http://porpax.bio.miami.edu/~cmallery/255/255chem/255chemistry.htm http://porpax.bio.miami.edu/~cmallery/255/255chem/255chemistry.htm]</p>
<p>[http://library.thinkquest.org/20465/DNAstruct.html http://library.thinkquest.org/20465/DNAstruct.html]</p>
{{Laboratory Experiments}}
{{Laboratory Experiments}}

Latest revision as of 05:08, 21 January 2024

Objective

The objective of this experiment is to use the biomedical forensic techniques of extracting DNA, fingerprinting, identifying foreign substances, and blood typing to investigate a crime scene.

Overview

Biomedical forensic methods are used in criminal or civil cases. They are also used within the medical field to diagnose and treat diseases and injuries. Within criminal law, these techniques are used to identify suspects in criminal cases and to exclude individuals as suspects. DNA testing, in particular, is increasingly used to prove the innocence of people who have been wrongfully convicted of a crime.

In this lab, DNA extraction will be introduced using basic biochemical techniques for isolating, purifying, and digesting DNA molecules. A toxicology report will be completed by observing chemical reactions using simulated reagents. A simulation kit will be used for the blood typing tests.

DNA Extraction

Deoxyribonucleic acid (DNA) is found in almost all living organisms. In this lab, DNA will be extracted from plant cells in an introductory exercise to this lab.

Plant cells are surrounded by structures called a cell wall and plasma membrane that protect the cell. Inside the cell sits various units called organelles; DNA is located in an organelle called the nucleus.

The DNA extraction process can be divided into three major steps: breaking open the cell wall, destroying membranes within the cell, and precipitating the DNA out of the solution.

The first step in DNA extraction is to break the cell wall (cell lysis) to expose the DNA within the cell. Cell walls are rigid structures made of cellulose and thus require mechanical methods to break apart. In this experiment, DNA will be extracted from a strawberry that will be mashed manually.

The second step in DNA extraction is to destroy the plasma membrane within the cell. The cell's plasma membrane is made of phospholipid bilayers that are made of fat. To disrupt them, the mesh of fat molecules is broken up with a surfactant (soap). The structure of soap is similar to the structure of fat and grease and lowers the surface tension between two fluids when acting as a surfactant.

A soap molecule has two parts: a head and a tail. The head is polar and is attracted to water (hydrophilic), while the tail is nonpolar and attracted to oil and fat (hydrophobic). When soap molecules are in water, they group themselves into micelles that have a roughly spherical structure in which all the polar heads point outwards (toward water) and all the nonpolar tails point inwards at the center of the sphere (away from the water) (Figure 5).

Figure 5: Soap Micelle

The soap molecules effectively trap the fat molecules inside the micelles and dissolve the cell membranes. The soap molecules orient themselves so that their head associates with the tail of the phospholipid bilayer. In this way, the soap breaks up the bilayer molecule by molecule (Figure 6).

Figure 6: Phospholipid Bilayer

The last step in DNA extraction is precipitating the DNA. When the plasma membrane is successfully disrupted, the DNA is released from the cells into the solution along with protein molecules and other cellular miscellany. With the cell's contents mixed into a solution, the DNA can be separated from the rest of the contents. This process is called precipitation. Salt is used because it disrupts the structure of the proteins and carbohydrates found in the solution. Also, the salt provides a favorable environment to extract the DNA by contributing positively-charged sodium ions that neutralize the negative charge of DNA.

After adding salt and soap, the extracted DNA cannot be seen as it is too small to distinguish from the rest of the solution. To make the DNA visible, alcohol is added since it cannot dissolve the DNA. A translucent white substance will begin to form at the top: this is the DNA (Figure 7). Once the precipitate is thick enough, it can be spooled out. This simple procedure is a rough extraction process that needs further steps before the DNA can be successfully run on a gel for analysis. This analysis will not be performed in this lab.

Figure 7: Extracted DNA

Fingerprints

Fingerprints are unique patterns consisting of friction ridges (raised) and furrows (recessed) that appear on the pads of the fingers and thumbs and are often used for identification purposes in forensics. Prints from palms, toes, and feet are also unique, but these are used less often for identification so this guide focuses on prints from the fingers and thumbs.

The fingerprint pattern, such as the print left when an inked finger is pressed onto paper, is made from the friction ridges on that particular finger. Friction ridge patterns are grouped into three distinct types – loops, whorls, and arches – each with unique variations, depending on the shape and relationship of the ridges.

Loops are prints that recurve back on themselves to form a loop shape (Figure 8). Divided into radial loops (pointing toward the radius bone, or thumb) and ulnar loops (pointing toward the ulna bone, or pinky), loops account for approximately 60% of pattern types.

Figure 8: Loop Fingerprint

Whorls form circular or spiral patterns, like tiny whirlpools (Figure 9). There are four groups of whorls: plain (concentric circles), central pocket loop (a loop with a whorl at the end), double loop (two loops that create an S-like pattern) and accidental loop (irregularly shaped). Whorls make up about 35% of pattern types.

Figure 9: Whorl Fingerprint

Arches create a wave-like pattern and include plain arches and tented arches (Figure 10). Tented arches rise to a sharper point than plain arches. Arches make up about 5% of all pattern types.

Figure 10: Arch Fingerprint

Blood Typing

Even though all red blood cells are made of similar elements, not all blood cells are alike. There are eight different types of blood, each based on the presence or absence of three types of antigens (A, B, and Rh) (Figure 11). Generally, antigens induce an immune response to foreign substances in the body. The A and B antigens determine the type of blood while the Rh antigen determines if the blood is positive or negative with the Rhesus factor, which is associated with hemolytic diseases and incompatible blood transfusions.

  • The ABO Blood Group System
    • Group A has only the A antigen on red cells
    • Group B has only the B antigen on red cells
    • Group AB has both A and B antigens on red cells
    • Group O has neither A nor B antigens on red cells

Positive (+) blood types have the Rh factor and negative (-) blood types do not have the Rh factor.

Figure 11: Different Blood Types

Materials and Equipment

  • Strawberry
  • Non-iodized table salt (NaCl)
  • Hand soap (clear, unscented)
  • 95% isopropyl alcohol (0 °C)
  • Distilled water
  • Strainer
  • Plastic cups
  • Ziploc bag
  • Iron magnetic wand
  • Iron powder
  • Blood typing kit
  • Pipettes
  • Beaker
  • Test tubes
  • Microcentrifuge tubes
  • Baby wipes
  • Meter stick

Procedure

Watch the following video: Biomedical Forensics Part 1

Forensic Academy

Basic training in three forensic techniques will be completed before the crime scene investigation is conducted.

1. DNA Extraction

  1. Place 200 ml of distilled water in a beaker.
  2. Add 2 tsp (10 ml) of dish soap to the water.
  3. Stir in ¼ tsp of salt and mix until the salt dissolves. This is the extraction mixture.
  4. Place one strawberry into a Ziploc bag.
  5. Pour the extraction mixture into the bag with the strawberry.
  6. Remove as much air from the bag as possible and seal it closed.
  7. Mash the strawberry inside the bag manually. Crush any large pieces.
  8. Pour the resulting strawberry pulp and extraction mixture through a strainer and into a beaker.
  9. Use a plastic spoon to press the mashed bits of strawberry against the strainer forcing as much of the mixture as possible into the container.
  10. Pour the extraction mixture into a test tube. This will help isolate the DNA on the surface of the mixture.
  11. Add 1 tsp (5 ml) of the chilled isopropyl alcohol to the solution (request the isopropyl alcohol from a TA) and hold the mixture at eye level. Look for the separation of DNA that shows up as a white layer on the surface of the mixture.
  12. If the separation of DNA cannot immediately be seen, set the small container aside for a few minutes and examine it again later.

2. Fingerprinting

  1. Obtain a clean piece of Plexiglass and thoroughly clean it with a baby wipe.
  2. Apply a thumb onto the glass and put pressure onto the glass while holding the glass by its edges. Keep applying pressure for a few seconds.
  3. Hold the glass against the light to view the imprint to determine if it is visible. If not, clean the glass and repeat the process.
  4. Take the iron filament and spread a generous amount on the region of the imprint.
  5. Take the magnetic wand and slowly pull the top across the iron filament to extract the excess iron filament from the Plexiglass piece.
  6. Place the excess iron filament back into the container and seal the container.
  7. Closely observe the remaining fingerprint and identify the fingerprint type.
  8. Take a picture of the fingerprint on a white background.

3. Toxicology Report

  1. Obtain samples of the suspect drug from a TA.
  2. Separate the drug sample into five microcentrifuge tubes and separately label the microcentrifuge tubes A, B, C, D, or E.
  3. Obtain the five drug reagents from a TA. The reagents test for the following drugs.

    Drug A: Cannabis
    Drug B: Heroin
    Drug C: MDMA (Ecstasy)
    Drug D: Cocaine
    Drug E: Lysergic Acid Diethylamide (LSD)
  4. Take the A-labelled microcentrifuge tube with the drug sample and place five droplets of the cannabis reagent.
  5. Observe the test tube. If a reaction is observed from adding the reagent, the tube contains that drug. The microcentrifuge tube might need a quick shake to catalyze the reaction.
  6. Repeat Steps 4 and 5 for each of the drug reagents.


Congratulations, the Forensic Academy is completed and the graduates are certified to participate in the crime scene investigation!

Crime Scene Investigation

It was a warm and stormy night on September 5, 1975. The EG1004 office was quiet as one TA was pulling a late night shift to prepare the classroom for the next morning’s lab. There was a loud crash in the background. The room went dark. The next morning, there was police tape barring entry to the entire room. Someone had murdered Ruhit Roy. At the crime scene, police discovered a pool of blood, along with the suspected murder weapon – a shard of glass – near the body. When the investigation yielded no results, Ruhit’s murder was declared a cold case. Watch the following video: Biomedical Forensics Part 2

After many years, Ruhit’s case is finally being reopened. Watch the following video: Biomedical Forensics Part 3

Download the Suspect Information Sheet to get started on the investigation.

1. Biological Dimensional Analysis

  1. Obtain a yard stick and measure the length of a foot with shoes on.
  2. Measure the length of a hand from the tip of the middle finger to the bottom of the palm.
  3. Repeat Steps 1 and 2 for the additional members in the group.
  4. Calculate and compare the ratios of the hand length to foot length and determine the average foot to hand length ratio based on the data obtained.
  5. Using the average foot length to hand length ratio, approximate the hand length of the murderer using the 11 in footprint found at the crime scene. Record the estimated measurements of the killer (Figure 12).
Figure 12: An 11" Footprint Left at the Crime Scene

2. Toxicology Report

  1. To collect important information on the murder, the victim’s blood will be tested to determine the drug that killed him.
  2. Put a small sample of Roy’s blood into five microcentrifuge tubes. Use the drug reagents to determine which drug was injected into his bloodstream by the killer.
  3. Record the drug that was used on the victim.

Note: Please use caution when using the microcentrifuge tubes, as they can explode.

Watch the following video: Biomedical Forensics Part 4

3. Blood Typing

  1. Obtain the blood sample from the crime scene and from all 10 suspects and the victim along with their respective A, B, and Rh serum capsules.
  2. Using a pipette, administer three drops of Suspect 1’s blood into all three ovals of the test tray.
  3. Using a pipette, administer a one drop of the A serum into the oval labeled A, administer one drop of the B serum into the oval labeled B, and administer one drop of the Rh serum into the oval labeled Rh. Wash the pipette out with water after administering each serum and between each suspect to prevent cross-contamination.
  4. If the blood sample exhibits a reaction to the indicator (bubbles or becomes cloudy), then the blood type matches that indicator type. If the liquid appears to be more diluted (more watery or thin), then the blood type does not match that indicator type. For example, if a precipitate forms for A and Rh for a blood sample, then it is A+. If a precipitate forms for A and B, but not Rh, that blood type is AB- If the blood samples do not react to any serum, then that blood type is O-.
  5. If a precipitate forms for A and B, but not Rh, that person’s blood type is AB-.
  6. Repeat Steps 2-5 for each suspect. Do not mix up the serums as that will determine the wrong blood type.
  7. The blood found at the scene of the crime was A-. Test Roy’s blood to make sure that the blood at the scene is the killer’s blood and not Roy’s blood.
  8. Add the blood types of each person to the Suspect Information Sheet to determine if any suspect’s blood type matches the blood type found at the crime scene.

4. Fingerprint Test

  1. Carefully obtain the murder weapon (the shard of glass).
  2. Carefully apply the iron filament at the rim of the glass shard, trying not to waste filament.
  3. Using the magnetic wand, carefully remove the excess filament.
  4. Fingerprint residue should be left behind after removing the excess filament; if not, repeat Steps 2 and 3.
  5. Observe the fingerprint, noting its features and type. Determine which person it corresponds to in the Suspect Information Sheet.

Using all the information gathered in the analyses, use the data provided in the Suspect Information Sheet to determine who the killer is. Tell a TA who the alleged killer is.

Assignment

There is no lab report for Lab 11.

Team PowerPoint Presentation

Follow the presentation guidelines laid out in the EG1004 Lab Presentation Format in the Technical Presentations section of the manual. When preparing the presentation, consider the following points.

  • Rely heavily on graphics and pictures
  • Make sure the experimental work is described simply and thoroughly
  • Discuss the real-life application of DNA extraction
  • Demonstrate clear understanding of each procedural step carried out and why it worked

References

http://www.vernier.com

http://www.invitrogen.com

http://en.wikipedia.org