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, test the moisture of a soil sample, and perform blood typing and gel electrophoresis. The DNA will be extracted using the basic biochemical techniques for isolating, purifying, and digesting DNA molecules. The moisture test will be completed using an Arduino moisture sensor. A blood typing kit will be used for the blood tests. While these tests are performed, the gel electrophoresis will be run simultaneously.
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.
In this experiment, a goal 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; they are made 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
 
Positive (+) blood types have the Rh factor and negative (-) blood types do not have the Rh factor.
 
[[Image:Lab_biomed_1.PNG|thumb|500px|frame|center|Figure 11: Different Blood Types]]


= Materials and Equipment =
= 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>
* Fruit sample
* Non-iodized table salt (NaCl)
* Hand soap (clear, unscented)
* 95% isopropyl alcohol (0 &deg;C)
* Distilled water
* Strainer
* Plastic cups
* Ziploc bag
* Lambda DNA
* Dye
* Variable micropipette and tips
* Incubator
* Microcentrifuge tube
* Precast agarose gel
* Electrophoresis system
* Bioimaging system
* DNA sample containers
* Disposable pipets
* Iron Magic Wand
* Iron powder
* Arduino software
* Arduino moisture sensor
* Blood typing kit
** Milk, water, vinegar, dye


<br style="clear: both;" />
*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
<!--*Plexiglass pieces-->
*Baby wipes
*Meter stick


= Procedure =
= Procedure =
NOTE: Some biological experiments require a lot of wait time. To maximize time, many experiments of this nature are usually performed simultaneously. In this lab, the multitasking technique that is used by professional scientists will be used. Please read the procedures carefully when jumping between different experimental procedures.
 
:: <h3>Preparation of Sample for Restriction</h3>
Watch the following video: [https://drive.google.com/file/d/18f1tHzFJg5bnU5ky9-HWcFqvm9FePp_K/view?usp=sharing Biomedical Forensics Part 1]
{| class="wikitable" style="float: right; text-align: center;"
 
|+ Summary of reagent amounts for DNA restriction.
== Forensic Academy ==
!Tube!!Lambda DNA!!Restriction Buffer!!BamH1!!Distilled H<sub>2</sub>O
 
!style="background-color: black;"|
Basic training in three forensic techniques will be completed before the crime scene investigation is conducted.
!Dye
 
|-
=== 1. DNA Extraction  ===
|1||4 &micro;L||5 &micro;L||2 &micro;L||8 &micro;L
 
|style="background-color: black;"|
# Place 200 ml of distilled water in a beaker.
|2 &micro;L
# Add 2 tsp (10 ml) of dish soap to the water.
|-
# Stir in ¼ tsp of salt and mix until the salt dissolves. This is the extraction mixture.
|Control||4 &micro;L||5 &micro;L||0 &micro;L||10 &micro;L
# Place one strawberry into a Ziploc bag.
|style="background-color: black;"|
# Pour the extraction mixture into the bag with the strawberry.
|1 &micro;L
# Remove as much air from the bag as possible and seal it closed.
|}
# Mash the strawberry inside the bag manually. Crush any large pieces.
# Load the micropipette with a tip and obtain four &micro;L of the lambda DNA using the micropipette.
# Pour the resulting strawberry pulp and extraction mixture through a strainer and into a beaker.
# Pipette it into a microcentrifuge tube and dispose of the micropipette tip. Dispose of the tip after each use.
# 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.
# Reload the micropipette with another tip and obtain five &micro;L of the restriction buffer. Dispose of the tip.
# Pour the extraction mixture into a test tube. This will help isolate the DNA on the surface of the mixture.
# Get another tip and get two &micro;L of BamH1, which is the restriction enzyme used to cut the DNA. Dispose of the tip.
# 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.
# Add eight &micro;L of distilled water. After this is done, call the TA.
# If the separation of DNA cannot immediately be seen, set the small container aside for a few minutes and  examine it again later.
#: A TA will oversee the rest of the procedure. As step 6 is occurring, proceed to the DNA extraction portion at step 16.
 
#: <h3> Restriction of DNA Sample </h3>
=== 2. Fingerprinting ===
# Place the microcentrifuge tube into the incubator. It should be set to 37 &deg;C. The sample will incubate for 30 minutes.
 
# Add two &micro;L of dye. This will show the DNA as it runs through the gel.
# Obtain a clean piece of Plexiglass and thoroughly clean it with a baby wipe.
#: <h3> Gel Electrophoresis </h3>
# 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.
# Prepare the electrophoresis gel when there are 15 minutes left for the incubation.
# 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.
## Plug the power base in.
# Take the iron filament and spread a generous amount on the region of the imprint.
## Open the package containing the gel.
# Take the magnetic wand  and slowly pull the top across the iron filament to extract the excess iron filament from the Plexiglass piece.
## Insert the gel, right edge first, and press firmly at the top and bottom to seat the gel in the base. A snap will be heard when it is in place. A steady red light will appear.
# Place the excess iron filament back into the container and seal the container.
## Press and hold either button until the red light turns into a flashing green light. This indicates that the two-minute pre-run of the gel has started. At the end of the pre-run, the flashing green light will change to a flashing red light and the base will rapidly beep.
# Closely observe the remaining fingerprint and identify the fingerprint type.
# Press and release either button to stop the beeping. One more beep will be heard. The light will change from a flashing red to a steady red light.
# Take a picture of the fingerprint on a white background.
# Remove the comb from the gel by pulling it straight up from both sides and remove any excess fluid using a pipette.
 
# Load the samples in 20 &micro;L volumes into the wells. Load 20 &micro;L of distilled water into any remaining empty wells.
=== 3. Toxicology Report ===
# Press and release the 30-minute button to start the 30-minute electrophoresis run. The light will change to a steady green light.
 
# Wait 30 minutes for the run to complete. The light will flash red and there will be a rapid beeping.
# Obtain samples of the suspect drug from a TA.
#: While waiting for the gel electrophoresis to complete, proceed to step 28.
# Separate the drug sample into five microcentrifuge tubes and separately label the microcentrifuge tubes A, B, C, D, or E.
# Press and release either button to stop the beeping and the light will turn to a steady red light.
# Obtain the five drug reagents from a TA. The reagents test for the following drugs.
# Remove the gel from the base and analyze the results using a UV transilluminator.
#: <br>
#: <h3> DNA Extraction </h3>
#: Drug A: Cannabis
#: Contaminants such as sugars remain in the DNA collected from the fruit sample. Typically, to properly run it through the electrophoresis gel and get results, it must be sized down considerably and thoroughly rinsed to get rid of the excess. Instead, Lambda DNA is used because it is already prepared and able to run in the electrophoresis gel.
#: Drug B: Heroin
# Obtain a fruit sample that is about two inches wide and put it in the Ziploc bag provided.
#: Drug C: MDMA (Ecstasy)
# Close the bag so there is as little air as possible inside.
#: Drug D: Cocaine
# Mash the sample gently by hans. Be careful not to burst the bag. After about five minutes, the fruit sample will be transformed into a creamy paste. This process is known as homogenization.
#: Drug E: Lysergic Acid Diethylamide (LSD)
# Prepare the buffer solution while the homogenization of the fruit sample is occurring.
# Take the A-labelled microcentrifuge tube with the drug sample and place five droplets of the cannabis reagent.
## Fill a cup &frac14;-way with distilled water.
# 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.
## Add one teaspoon of table salt.
# Repeat Steps 4 and 5 for each of the drug reagents.
## Mix the solution until the salt dissolves in the water.
 
## Add two teaspoons of soap.
 
## Stir gently with the spoon so that it does not foam. Keep stirring until the texture of the solution is even.
<b>Congratulations, the Forensic Academy is completed and the graduates are certified to participate in the crime scene investigation!</b>
# Pour the prepared buffer solution into the Ziploc bag and close it. Make sure that there is no trapped air in the bag.
 
# Mix the smashed fruit and the buffer solution gently in the bag. Do it slowly. It is important that it does not foam a lot.
== Crime Scene Investigation ==
# Let the mixture sit for about five minutes. If it has foamed, allow the foam to go away during this time. By letting the mixture stay still, the foam will disappear.
 
# Filter the solution by using another clear plastic cup. Hold the strainer on top of the empty cup while carefully pouring out the contents of the Ziploc bag. Make sure it does not foam. Pour slowly. Occasionally shake the strainer to make the liquid filter through. There is a lot of debris.
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]
# Add ice-cold 95% isopropyl alcohol to the filtered solution by pouring the alcohol against the wall of the cup. Do not mix the alcohol with the solution; it should float on top. Alcohol dissolves within water, but it can float if it is poured slowly against the side of the container because it is less dense than water. Pour the alcohol until the total volume reaches &frac34; of the cup’s volume. After about one minute, threads of DNA will form into translucent gel-like globs at the interface of the filtered solution and the alcohol.
 
# Collect the DNA by spooling it with a paperclip.
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]
# Split the spooled DNA into two sample containers. Label one S for sugars and the other P for proteins.
 
# Return back to step 7.
<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>
#: <h3> Chemical Tests for Biological Substances </h3>
 
# Obtain the Benedict's and Biuret reagents.
=== 1. Biological Dimensional Analysis ===
# Note the original color of the Benedict's and Biuret reagents as well as the color of the original spooled DNA sample.
# Obtain a yard stick and measure the length of a foot with shoes on.
# Use a transfer pipet to place three drops of Benedict's reagent onto the spooled DNA in the sample container marked S and note any color change.
# Measure the length of a hand from the tip of the middle finger to the bottom of the palm.
# Repeat step 29 with the Biuret reagent in the container marked P and note color change.
# Repeat Steps 1 and 2 for the additional members in the group.
# Clean up all materials. Throw out the extracted DNA and go back and complete steps 14 and 15.
# 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).
 
[[Image:Foot.png|thumb|600px|center|Figure 12: An 11" Footprint Left at the Crime Scene]]
 
=== 2. Toxicology Report ===
 
# 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.
 
<font color="red"><b>'''Note: Please use caution when using the microcentrifuge tubes, as they can explode.'''</b></font>
 
Watch the following video: [https://drive.google.com/file/d/1yCpCj2oNPDNIy-90OfRXMC4ZazPpiMRk/view?usp=sharing Biomedical Forensics Part 4]
 
=== 3. Blood Typing ===
 
# 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.
 
=== 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:
Author a presentation using (nearly) all images. The following areas are where text is allowed:
* Rely heavily on graphics and pictures.
* Title slide
* Make sure the Experimental Work is described simply and thoroughly.
* Overview slide
* Discuss the real-life application of DNA sequencing.
* Materials slide
* Demonstrate clear understanding of each procedural step carried out and why it worked.
* Captions for figures, tables, and equations
* Suspect list/table
* Data tables
* References to suspect names
'''Text is allowed for slide titles'''
 
{{Labs:Team Presentation}}
 
* 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 =
= 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 19:39, 18 April 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

Author a presentation using (nearly) all images. The following areas are where text is allowed:

  • Title slide
  • Overview slide
  • Materials slide
  • Captions for figures, tables, and equations
  • Suspect list/table
  • Data tables
  • References to suspect names

Text is allowed for slide titles

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