Link to our lab as a pdf - College of Science - Marshall University

An Identity Crisis 

A Lab on DNA Typing 

The Mystery of Lyle and Louise 1 

rev. 3/4/2008

The Mystery of Lyle and Louise: 

Identity Crisis: 

A Lab on DNA Analysis 

National Science Education Standards: 

Unifying concepts and processes 

Science as inquiry 

Life science 

Systems, order, and organization 

Evidence, models, and explanation 

Change, constancy, and measurement 

Abilities necessary to do scientific inquiry 

Understanding about scientific inquiry 

The Cell 

Molecular basis of heredity 

Science and technology Understanding about science and technology 

Science in personal and 

social perspectives 

Science and technology in local, national, and global 

challenges 

History and nature of science 

Science as a human endeavor 

Nature of scientific knowledge 

Visit Us at: 

www.LyleAndLouise.com 

Cover and Interior Art by Phil Morrissey 

“Forensic DNA Technology” and “PCR Technology” by Jan Sikorsky, PhD, and Julie Sikorsky, MS 

Edited by M. Joseph Hughes, MS 

Special thanks goes to the Integrated Science and Technology Department at Marshall University for their help in developing this suite. 

Copyright © 2006-2008 Vandalia Research, Inc. 

All Rights Reserved. 

www.vandaliaresearch.com 

2 

The Mystery of Lyle and Louise

Table of Contents 

Introduction to Identity Crisis. . . . . . . . . . . . . . . . . . . . . . . . . . 5 

Teacher’s Notes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 

Teaching Timeline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 

Solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 

Forensic DNA Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 

PCR Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 

The Investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 

The Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22 

The People Involved. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 

Pre-Lab Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 

Lab Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27 

Data Collection and Calculations . . . . . . . . . . . . . . . . . . . . . . .29 

Post-Lab Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30 

Mock Trial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31 

The Mystery of Lyle and Louise 3

4 


Introduction to Identity Crisis 

WELCOME to Identity Crisis, a DNA Typing lab in the Mystery of Lyle and 

Louise. A brutal murder case is unfolding in a small Appalachian town. 

Already the case spans two crime scenes and five people are dead. However, 

two women, one at each crime scene, have been identified as Louise Mondelo. 

Your students, as the forensic 

investigators responsible for 

the DNA evidence, must positively 

identify Louise Mondelo 

and also analyze the DNA 

evidence for clues that might 

help crack the case. 

In this lab, students will learn 

about DNA Typing and related 

technologies. These include 

the polymerase chain 

reaction (PCR), a method for 

amplifying small samples of 

DNA, and electrophoresis, a 

method for separating bands 

within the PCR product. An 

introduction to short tandem 

repeat DNA analysis is also 

covered in this booklet. 

In addition to reading information provided about the case, students will test 

DNA samples from people and objects, compare them to each other, and make 

assessments of the probability that samples came from the same source. Once 

the lab results have been analyzed, students may host a mock trial to hold a chosen 

suspect accountable for their actions. 

Teacher’s notes can be found at the beginning of the manual and copies may be 

freely made of all materials for your students. 


6 


These notes are provided to assist in the preparation 

and execution of the laboratory experiment. 

A solutions key for the pre- and post-lab 

questions can be found on the reverse. 

Supplies 

First, inventory the supplies included in the lab kit. 

Supplies have been provided for up to six groups of 

students. 

• DNA for Known Gels (3 sets) 

• DNA for Evidence Gels (3 sets) 

• Agarose Gels (6 bottles) 

• 20x BlueVis Buffer (2 bottles) 

• BlueVis Gel Stain (1 bottle) 

• InstaStain Sheets (6 sheets) 

Refrigerate the entire contents of the kit until ready 

to use. 

Other Supplies and Equipment Required 

• Lab gloves 

• Distilled water 

• 1 to 4 L capacity bottle 

• Microwave 

• 5 mL Pipettes and pipettor 

• Gel apparatus (6) and power supply 

• 10 μL Pipettor and Pipette tips 

• OPTIONAL: Staining trays 

Running Buffer Preparation 

The 20x BlueVis Buffer running buffer must be diluted 

to 1x before being used in the lab. Each 200 

mL bottle of buffer will make 4 L of the solution 

used in this lab. 

Shake the Blue Vis Buffer well before use. To make 

1 L of 1x BlueVis Buffer add 50 mL of the 20x stock 

solution to a 1 L capacity bottle, and bring the total 

volume up to 1 L with distilled water. 

Safety Precautions 

1. 

2. 

3. 

4. 

5. 

Teacher’s Notes 

Molten agarose can scald and it must be handled 

with care 

Serious or lethal electrical shock can result if 

the gel apparatus is used improperly. 

Remove caps from bottles placed in the microwave 

to avoid explosion and serious injury. 

BlueVis buffer and Gel Stain will stain skin and 

clothing; wear gloves while handling. 

Review MSDS’s found on the Mystery of Lyle 

and Louise website. 

http://www.lyleandlouise.com/msds 

Pipetting Practice 

Inexperienced students should practice pipetting 

before performing this lab. One suggestion is to use 

a small piece of waxed paper and practice picking 

up and delivering small volumes of colored water. 

Students should hold the pipettor as vertical as 

possible. Without precise pipetting, experimental 

results may not be accurate. 

Notes for the Gel 

The kit includes enough samples for 3 groups to 

analyze known suspects and victims (Gel A) and another 

3 groups to analyze the unknowns and evidence 

(Gel B). DNA labels are color-coded to keep 

them separate in the lab; DNA that should be run on 

Gel A are in blue, DNA for Gel B are in red. Ladder 

DNA, which must be run on both gels, has a white 

cap. 

The BlueVis Gel Stain is the same solution as the 

20x BlueVis TBE running buffer, but is bottled separately 

to prevent confusion. If more of the stain is 

needed, remaining 20X BlueVis Buffer can be used 

with no problem. Gels thicker than 1 cm have poor 

contrast. Use less than the 80 mL of agarose provided 

if you have small gel boxes. Note: BlueVis will 

not stain DNA if it is overheated. Don’t remelt agarose 

after adding BlueVis. 


Teaching Timeline 

Groundwork 

Before performing any of the lab exercises in the Lyle and Louise suite, cover the investigation and 

background story to give students a framework for the work they are about to do. This only needs to be 

covered once for all of the modules. 

Day 1: Cover material in the Forensic DNA Technology Background 

Explain how DNA is collected at crime scenes, why DNA amplification is necessary, and how PCR amplification 

works. If you have long class periods (over an hour) save explaining how DNA migrates for the 

portion of the lab when students are watching the DNA migrate in the gel. 

Days 2-4: Lab Procedure 

The DNA Typing kit is a flexible kit, with several stopping points for teachers who have short class periods 

or slow gel electrophoresis apparatus. The following instructions will assist you in determining how 

to organize this kit for your class. 

Melting and Casting Gels - This can be done in the early part of the class period, and for classes that 

are long enough, you can start to run the gels. Otherwise, store the gels in a ziploc bag overnight in the 

refrigerator. 

Running the Gel - Start by adding the BlueVis running buffer to the electrophoresis chamber. This 

should be done when you plan to start running the gel - don’t let the gel set overnight in the buffer. For 

classes with slower electrophoresis boxes (where the gel will take longer than the class period to run), 

we have included optional InstaStain sheets to resolve the DNA so that the gel can be stored overnight. 

Analyzing the Gel in Real-time - While the gel is running, and for several hours after it has run, you can 

view the bands with the BlueVis dye present. The BlueVis dye will fade away eventually, and cannot be 

stored overnight. 

Analyzing the Gel with InstaStain - If you need to store the gel overnight, or wish to resolve the bands 

more for photodocumentation purposes, you can restain the gel with the included InstaStain sheets. 

Follow the procedure below: 

1. Place the gel in a shallow plastic container. Fill the container with distilled water. 

2. Float the InstaStain sheet, blue side down, on the water. 

3. Allow the gel to stain and destain overnight. 

8 


Glossary 

Agarose: A molecule purified from seaweed that is 

used for the electrophoretic separation of nucleic 

acids. 

Allele: One of two or more alternative forms of a 

gene that exist at a specific gene location on a chromosome. 

Base: A letter in the DNA alphabet, either adenine 

(A), thymine (T), guanine (G), or cytosine (C). 

Band: A discrete length of DNA that is visualized on 

a gel when the DNA in the gel is stained. 

Buffer: A solution made of acids and their conjugate 

bases that is used to maintain the pH of a system. 

Chromosome: A linear strand of DNA and proteins 

in the nucleus of a cell. Chromosomes carry genes 

and function in the transmission of hereditary information. 

Chromosomes are in pairs, one set coming 

from each parent. 

Comb: A plastic piece shaped like a comb that leaves 

wells in agarose gels. 

DNA: Deoxyribonucleic acid (DNA) is a doublestranded, 

helical nucleic acid molecule that is the 

carrier of genetic information. 

Gene: A discrete unit of hereditary information 

that is located on the chromosomes and consists of 

DNA. Genes can have many alleles. 

Heterozygous: Having two different alleles for a 

given trait, one from each parent. 

Homozygous: Having the same allele twice for a 

given trait. 

Load: Placing the DNA sample in the well of the 

agarose gel with the pipet. 

PCR: The Polymerase Chain Reaction, a technique 

used to amplify regions within small amounts of 

DNA by copying those regions many times. 

Pipette: A device used to pick up and deliver a precisely 

measured amount of liquid. 

Tips: A small, disposable plastic piece which fits on 

a pipet. The tips are changed to prevent samples 

from cross-contamination. 

Well: Small rectangular hole in agarose gel left by 

comb. Each DNA sample is loaded in a well. 

Electrophoresis: When an agarose gel is placed in 

buffer, DNA samples are placed in wells, and electrical 

current is passed through the gel, the electrical 

field pulls the DNA through the gel. Smaller molecules 

pass through easily and move toward the other 

end faster, thus sorting the DNA sample by size. 

Forensic: Relating to the application of scientific 

knowledge to legal cases. From Latin forensis for 

“belonging to the forum,” ancient Rome’s site for 

public debate and currently meaning pertaining 

to the courts. Thus, forensic testimony or forensic 

medicine are used to assist the court or the attorneys 

in legal matters, including trials. 

Gel: A gelatin-like material formed from long molecules 

(polymers) of agarose which trap liquid within 

a matrix. 

12 


Each individual, with the exception of identical 

twins, has a unique array of genetic information 

within their deoxyribonucleic acid (DNA). This 

information is the same in each nucleated cell in 

the body and acts as a “genetic blueprint” containing 

the instructions to build living organisms. This 

blueprint specifies physical characteristics such as 

eye color, height, and blood type. 

In the early 1950s, two scientists, James Watson and 

Francis Crick, determined the 3-dimensional structure 

of the DNA molecule. Watson and Crick knew 

that DNA was made up of four building blocks called 

nucleotides. Each nucleotide contains a sugar, a 

phosphate and one of four different bases. Watson 

and Crick knew the base Adenine (A) is present in 

the same amount as the base Thymine (T) while the 

base Cytosine (C) is present in the same amount as 

the base Guanine (G). They also had an X-ray picture 

of the crystalline structure of DNA by another 

scientist, Rosalyn Franklin, which showed DNA has 

a helical structure, like a corkscrew. Watson and 

Crick built a DNA model with two strands of chemically 

linked nucleotides that twisted around each 

other to form a ladder-like structure called a double 

helix. A negatively charged phosphate/sugar backbone 

forms the legs of the ladder. Pairs of nucleotides, 

one on either side of the ladder, face the center 

of the helix and are linked with hydrogen bonds 

to form the ladder’s rungs. T only fits with A and 

C can only fit with G. Because of this paring, each 

strand contains the information necessary to build 

the other strand, serving as a template. 

The bases A, T, G, and C are the letters in the DNA 

alphabet that spell the words our bodies understand. 

Genes are different words produced by this 

DNA alphabet that determine the characteristics of 

each individual. Each person has two complete sets 

of DNA split up into 23 pairs of chromosomes, one 

from their mother and the other from their father. 

Each parent can give us the same version, or allele, 

of a gene (homozygous), or we can receive two different 

versions (heterozygous). These genes are 

then used by our bodies to build a variety of proteins 

that are directly responsible for the physical 

Forensic DNA Technology 

traits we possess. This 

is why people often exhibit 

a mixture of their 

parent’s physical traits. 

Within the entire human 

population, however, 

there is less than 0.1% 

difference within our 

genetic makeups; that 

is, 99.9% of your DNA sequence 

is identical to all 

of the other people in the 

world. The fraction of a 

percent that is different 

is enough to produce all 

of the genetic variation 

exhibited between individuals. 

Not all DNA base combinations, 

however, produce 

genes that translate 

into proteins. Because 

these portions of DNA 

do not affect the physical properties of an individual, 

they are able to mutate with no ill effects, and 

transfer the changes to offspring. Therefore, this 

so-called “junk DNA” has a large degree of variability. 

By exploiting this variability, forensic analysts 

can purify DNA found at crime scenes, generate a 

unique DNA profile for a sample, and compare that 

profile to profiles from suspects or other individuals. 

Ultimately, the forensic analyst can determine 

a probability that two DNA samples came from the 

same individual. 

Forensic DNA analysis has had a short but exciting 

history. In 1984, Sir Alec Jefferys used a technique 

called Restriction Fragment Length Polymorphism 

(RFLP) analysis in the first legal case. Restriction 

enzymes are proteins that find all instances of 

short, specific DNA sequences, called cut sites, and 

cut the DNA molecule at the sequence. These enzymes 

are chosen such that they cut DNA at locations 

common to many people. Mutations in DNA, 

however, may create new cut sites or change the 


PCR Technology 

PCR is the cornerstone of current forensic DNA analysis as well as many other biological disciplines, 

such as drug discovery and disease diagnosis. PCR was invented in the mid-1980s by Dr. Kary Mullis, 

who was awarded the Nobel Prize in Chemistry in 1993 for this revolutionary breakthrough. PCR is 

like a biological photocopier. This sensitive technique is capable of producing millions, even billions 

of copies of DNA from just a few input strands (templates) of DNA. Once copied a billion-fold, this 

small amount of original DNA can now be visualized using gel electrophoresis in cases where, prior 

to this process, it was undetectable. 

Ingredients of PCR: 

Template DNA: the input DNA to be copied (DNA 

from the crime scene). 

This DNA is obtained by extracting it from 

a cell’s nucleus and separating it away from 

protein and other cellular components. 

Primers (forward and reverse): bookends used to 

define the region of junk DNA to be copied 

Primers are short pieces of DNA synthesized 

in a laboratory that bind to DNA adjacent to 

the polymorphic or variable region. Each 

section of DNA used for amplification requires 

two primers, a forward and reverse, to 

define the start and stop point for the reaction. 

dNTPs: the building blocks of DNA 

PCR products are made from the same building 

blocks as DNA. dNTPs (deoxynucleotide 

triphosphates) is a collective term for any or 

all bases. 

PCR Buffer: the solution in which PCR occurs 

A solution containing precise concentrations 

of salts and Magnesium that facilitate an ideal 

environment for PCR to occur. 

Taq Polymerase: the enzyme responsible for making 

the copies of DNA 

This enzyme was isolated from a bacterium 

(Thermus aquaticus) originally found in hot 

springs. In order to survive in this hostile 

environment, this bacterium evolved a specialized 

enzyme to replicate DNA at temperatures 

that would normally prevent enzymatic 

activity. This heat-stable enzyme is crucial 

to the PCR technique, as it assembles the raw 

components into the desired PCR products 

under the higher temperatures needed for 

the reaction. 

Three Stages of PCR: 

PCR is conducted in commercially available instruments 

called thermal cyclers. These instruments 

heat and cool the reaction tubes that contain 

the reaction components. Three steps are 

repeated approximately 30 times (called cycles), 

which results in the replication of billions of 

copies of the targeted DNA sequence. 

1. Denaturation: Temperature greater than 

90°C. The hydrogen bonds of the doublestranded 

DNA ladder are broken, allowing 

the two strands to separate. 

2. Annealing: Temperatures between 50°C to 

65°C. The primers sit down on the DNA, 

outside the targeted region to be amplified, 

binding by reforming hydrogen bonds to the 

specific complementary sequences flanking 

the target region, thus defining the region 

to be copied. 

3. Extension (Elongation): Temperature of 

approximately 72°C. Taq polymerase is activated 

and incorporates the free dNTPs into 

the area between the primers. 

14 



sequence at that site, preventing a cut. The lengths 

of DNA between the cut sites vary because of these 

mutations. The varying lengths of DNA fragments 

reflect the uniqueness of the DNA sample, and are 

compared to fragment size patterns generated from 

other DNA samples. RFLP analysis requires a large 

amount of intact DNA to obtain a meaningful result. 

While some crime scenes have much DNA-containing 

evidence, most contain only a small amount of 

material to work with, limiting the usefulness of 

RFLP analysis. 

The ability of forensic analysts to copy small 

amounts of DNA into larger quantities is critical 

when faced with crime scenes with little biological 

evidence – a cash register that has only been 

touched by a suspect, for example. It is also likely 

that some or all of the biological material found at a 

crime scene has been exposed to environment degradation 

in the form of weather, time, or the sun’s 

UV rays. The polymerase chain reaction (PCR) is 

a laboratory technique that mimics the biological 

process by which DNA replicates within a cell. 

Unlike RFLP which cuts DNA apart to produce an 

identifying pattern, PCR makes many copies of a selected 

region of junk DNA that has variable length 

within the population. The identifying pattern is 

created when the collective lengths of eight to sixteen 

of these regions, called short tandem repeats 

(STR), are compared. 

Figure 1. Possible genotypes of offspring given the genotypes of the 

mother and father. 

STRs used in DNA typing are located on different 

chromosomes, and therefore follow Mendel’s laws 

of segregation and independent assortment. These 

laws assert that we receive exactly one STR allele 

from each parent, and that receiving any given allele 

in no way influences the alleles received for 

other STRs. STRs can be used for paternity testing, 

because half of the offspring’s alleles must come 

from one parent and the other half from the other 

parent. 

Figure 1 shows a mother with the 2 and 3 repeat alleles 

of an STR locus, while the father has the 3 and 

6 repeat alleles. The Punnett square predicts the 

possible allele combinations for their children. The 

father can contribute either the 3- or the 6-repeat 

allele to his offspring, while the mother can contribute 

either the 2- or the 3-repeat allele. They 

could have four children with completely different 

STR genotypes. By chance alone, they could also 

have four children with the same genotype. However, 

they cannot have a child with a new allele, unless 

there was a mutation, which is very rare. 

Electrophoresis is used to separate DNA fragments 

by size. Electrophoresis takes advantage of fact that 

there are electrical charges carried by the phosphate 

backbone of the DNA molecule. Since DNA 

fragments are negatively charged, they migrate toward 

the positive pole when placed in an electric 

field. When DNA is forced to move through a separation 

gel matrix, fragments are sorted based on 

size because the gel prevents larger fragments from 

travelling as quickly as smaller fragments. The 

most common separation matrices used are agarose 

and acrylamide gels. Agarose gels are made 

from a component extracted from seaweed which 

is dissolved in a heated solution. When the solution 

cools, microscopic pores are formed that act 

as a molecular sieve through which DNA molecules 

pass. 

Think of the separation matrix as a kelp forest dense 

with fronds but having pockets of space in between. 

These spaces are much like the pores in the gel. Due 

to their size, small fish have an easier time navigat- 



stabilizes the gel’s pH. This same solution is also 

used to submerge the gel, providing an electrically 

and chemically uniform medium for the process. 

Since the phosphate backbone of DNA only has a 

negative charge in neutral to basic solutions, the 

solution is pH buffered to prevent it from becoming 

acidic, which would cause the DNA to reverse direction 

and move toward the negative pole. 

Figure 2: gel visualization of genotypes. 

ing through the plants than do the larger fish. Both 

eventually get through, but the small one travels 

faster. Applying this analogy to a gel matrix, the 

DNA travels through a gel, and just as in the seaweed 

forest, smaller fragments travel faster than 

the larger. When, after a certain period of time, the 

electrical force that moves the DNA through the 

matrix is removed, the newly spread-out DNA fragments 

stop moving. Their position in the gel is a 

direct relation to their size and they can be stained 

and their sizes compared to one another. 

Figure 2 is a schematic of a stained gel of each of 

the four possible genotypes of children from the 

mother and father in Figure 1. Notice how there is 

only one band for the homozygous genotype, C, because 

both the mother and father contributed the 

same allele. When visualized, this band should be 

darker than the other bands because there is twice 

as much DNA in that area as in the other bands. 

In electrophoresis, the solution used to create an 

agarose gel contains a salt dissolved in water, normally 

either Tris-acetate-EDTA (TAE) or Tris-borate- 

EDTA (TBE), that promotes electrical current and 

16 

Following separation by electrophoresis, fragments 

can be visualized by using several methods 

that directly bind to the DNA molecule. The most 

common in an educational setting is to stain with 

methylene blue. After electrophoresis, the gel is 

soaked in a stain solution. The stain adheres to the 

DNA molecules, but is also absorbed into the pores 

of the gel. Because of this, the entire gel is blue until 

it is allowed to soak in clear water, which dilutes 

the stain absorbed into the gel; a process called destaining. 

The dye bound to the DNA molecules is 

not removed during de-staining, and may be seen 

with the naked eye. In commercial and research 

settings, ethidium bromide is the most common 

nucleic acid stain. However, it can only be visualized 

under UV light and is mutagenic, which makes 

it unsuitable for novices. In this lab, a proprietary 

formula called BlueVis is used. BlueVis has no special 

safety concerns and stains the DNA while it migrates 

on the gel, reducing the need for de-staining. 

Other visualization methods include radioactive 

labels and commercially available fluorescent dyes 

which can be incorporated during the PCR process. 

To confirm the size of each PCR product, DNA size 

ladders are run on the same gel as the unknown 

samples. These ladders contain a series of known 

purified DNA fragments corresponding to most of 

the known variant forms of each portion of target 

DNA; each variant form is called an allele. Therefore, 

these allelic ladders provide a means for determining 

the length, and thus the specific variant 

or allele type, of each amplified product for a given 

sample. Theoretically, a large number of alleles are 

possible for any given loci; however, generally only 

four to ten alleles are common within a population. 

The alleles lengths that are present on the ladder, 



are the common ones. Those rare alleles in the 

population are collectively known as ‘off ladder’ alleles 

and are not included. A compilation of these 

STR comparisons from as may as 16 different, independent 

STR loci make up an individual’s genetic 

profile. 

Genetic profiles are created for both unknown 

samples from a crime scene and those that are obtained 

directly from a person who may be a suspect. 

Those taken from people, whether they are a suspect, 

victim, or any other individual, are referred 

to as “knowns.” They are used to compare known 

profiles against evidence profiles. An individual is 

excluded as a suspect when their profile does not 

exactly match the questioned sample’s profile; they 

are included only when the fragment sizes are identical 

at every STR locus in the DNA. 

illustrated a step forward in this rapidly evolving 

discipline. As technology and laboratory technique 

grow together, further innovations in the science of 

determining unique identity are assured. 

Additional Reading 

Butler, J.M. (2005) Forensic DNA Typing: Biology, 

Technology, and Genetics of STR Markers (2nd 

Ed). Elsevier Academic Press, New York. 

National Research Council (1996) Evaluation of Forensic 

DNA Evidence. National Academies Press, 

Washington D.C. 

STR DNA typing is an examination of the length of 

certain repetitions in a genome. In order to weigh 

the importance of each match, the final step in DNA 

typing is a statistical analysis. By sampling a large 

number of people in a particular population, researchers 

have determined allelic frequencies for 

the STR loci used in forensic DNA typing. By multiplying 

the population frequencies of the alleles 

present in a genotype, the probability of another 

person in a population having the same alleles at 

each loci can be determined. As the number of loci 

in the analysis increases, the probability of a random 

sample matching decreases geometrically. 

Thus, the probability is the result of both the rareness 

of the individual alleles, the distinctiveness of 

their combination, and the number of different STR 

loci compared. 

Siblings, however, will not share the same statistically 

random chance of receiving their alleles, since 

they can only receive the alleles their parents possess. 

Therefore, it may require testing of many additional 

STR loci to separate siblings using DNA typing. 

Further, identical twins share identical DNA 

and cannot be separated. 

DNA analysis has revolutionized forensic identification. 

Each of the technologies outlined here have 


18 


Nine days ago, during the night of a sudden 

summer thunderstorm, the Mondelo family 

car went over the side of Backbone Mountain and 

caught fire on impact. Three bodies were found in 

the wreckage; an adult woman, a teenage male, and 

a female child. All were burned beyond recognition. 

The three victims were identified as Louise 

Mondelo and her children Wally and Jan by personal 

effects that survived the fire,. 

Pictures of the scene were recorded, but, due to 

the rainstorm, the crash was initially believed to 

be simply a tragic accident and was not treated as 

a crime scene. When Lyle Mondelo could not be 

reached and was found to be missing, he became a 

possible suspect, and the wreckage was thoroughly 

processed. However, the scene was substantially 

disturbed, and some evidence was undoubtably 

lost. The bodies, as part of an ongoing criminal investigation, 

were kept in the county morgue. 

The Investigation 

The small town of Highland Park was shocked, since 

nothing this terrible had ever happened in the area. 

Tips from neighbors and friends poured into the 

police department, but none of the tips were eyewitness 

accounts or provided specific information 

regarding the car accident. Lyle was the likely suspect, 

but was nowhere to be found. An all points 

bulletin was issued for everyone to be on the lookout 

for Lyle Mondelo. He was presumed armed and 

dangerous and to be driving a missing blue 1993 

Ford Ranger with Tumbling Water Land Development 

Co. logos. Four days ago, Lyle Mondelo’s credit 

card was used to purchase gasoline and food at a 

gas station in Texas. 

When contacted, business associate John Wayne 

Gretzky told investigators that Lyle had been slipping 

into a deep depression because of trouble at 

their jointly owned business, Tumbling Water Land 

Development Company. Gretzky also hinted that 

there had been problems in the Mondelo family. At 



this time, investigators noticed that John had a large 

bite mark on his upper arm. When asked about the 

wound, Gretzky claimed to have been bit during a 

bar fight the night before and allowed the bite to 

be photographed. He was not held or charged with 

any crime. 

Background Investigation 

With no additional leads, policed launched a full investigation 

into the Mondelos. Louise Wilson and 

Lyle Mondelo had met at college while receiving 

Business Degrees in Management. They married in 

college and moved to Highland Park, Louise’s hometown, 

after graduation. The town was still ailing at 

the time, suffering from the shut down of the mines 

a little over a decade ago. Although at first Lyle 

thought their business prospects in the small town 

were poor, he soon discovered that money could be 

made developing land for the private lodges and ski 

resorts that employed most of the residents. 

After returning to Highland Park, Louise ran into 

her old High School sweet heart, John Wayne Gretzky. 

While talking to him, Louise learned that he was 

also a developer. Glad to see an old friend, and thinking 

that a favorable business relationship could develop, 

Louise asked John to meet with her and Lyle 

over dinner. Lyle and John soon became friends, 

and rather than compete for business against each 

other, the three decided to join together and start 

Tumbling Water Land Development Company. 

About a year after Tumbling Water was founded, 

Louise conceived her first child, Wally. Friends of 

the Mondelos said that Lyle suspected Louise and 

John of having an affair at the time, and the two 

nearly divorced. The couple, however, worked out 

their relationship with the help of a marriage counselor. 

20 

Tumbling Water became prosperous and was able 

to buy several hundred acres of land adjacent to 

Blackrock River, a prime recreational waterway. 

Soon thereafter, Louise had another child, Jan, and 

took leave from the office to work from home while 

she raised the two children. Friends say that Louise 

never really went back to Tumbling Water, even 

after the children were older and in school. Their 

friends also suggested that Lyle and Louise’s relationship 

was healthier with them not working together. 

Tumbling Waters’ lawyer told investigators that she 

began preparing bankruptcy papers for the company 

about a year ago; the ski resort was dragging out 

negotiations for a property purchase, and the company’s 

other business deals weren’t making enough 

profit to keep the business afloat. Soon after being 

asked to begin the bankruptcy filing, though, she 

said an unexpected deal was made to build a number 

of fishing cabins on the Blackrock River land. 

That was enough to keep the business going, and 

after that, Tumbling Water began making deals at 

a steady rate. 

A potentially related case recently touched on the 

Mondelos’ lives. Three weeks ago, a crystal methamphetamine 

lab was discovered in an abandoned 

camper on Tumbling Water land. Louise’s nephew, 

Mitch Wilson, and John Wayne’s brother, Larry 

Gretzky, were found in the lab and indicted for possession 

with intent to sell the 6 kilograms of meth 

found in the lab. Two days later they were both released 

on bond, posted by Lyle Mondelo and John 

Gretzky. Mitch and Larry gave no names of possible 

suppliers or dealers. 

Two weeks before the crash, Louise Mondelo filed 

for divorce. Friends say she told them that she suspected 

Lyle of being involved with drugs, but that 

the friends believed she was involved with John 

Wayne Gretzky again. Two days later after filing for 

divorce, Louise requested a peace bond against Lyle, 

stating that Lyle had harassed her and the children. 

Louise also told police that she was afraid that Lyle 

might try to take the children away from her. 

When attempting to contact Mitch Wilson and Larry 

Gretzky for questioning about the car accident, 

police discovered that they had both skipped town 

along with Larry’s girlfriend Mary Bradey. Authorities 

believed that their disappearance could be related 

to the accident, and they were described as 



possibly armed and dangerous in 

the warrant posted for their arrest. 

Two days ago, an abandoned blue 

Ford Ranger with out-of-state 

plates was found on a strip of 

New Mexico highway. The pickup 

was dirty and dusty, but investigators 

noticed a Tumbling Water 

Land Development Co. sign on 

the back tailgate. Forced entry 

was apparent. Upon access to the 

truck, investigators discovered 

several pieces of trace evidence 

and sent it to Highland Park for 

analysis. 

At the Scene 

The forest gets thicker and buildings less frequent 

the farther you drive along old Route 52. You see 

the sign for Tumbling Water Land Development 

Company and make the turn onto the gravel road 

cutting into the forest. Before long you see the other 

police vehicles, and Detective Murray is motioning 

you over to the side of the road. A small cabin, 

now surrounded by police tape is sheltered by trees 

here, and you see several other similar cabins further 

down the road. You hear the Blackrock River 

roaring not too far off behind the cabin. 

As you and your partner get out of the car, the hot 

July sun hits you. Detective Murray comes up, 

sweating in the heat. “Hey folks, welcome to the 

scene. Two bodies 

inside, both pretty 

bad off, and who 

knows how long 

they’ve been there. 

A Girl Scout found 

them about an hour 

and a half ago on a 

hiking trip. Gave 

her quite a fright; 

her troop leaders 

weren’t much better 

off. Anyway, we’ve got a problem. We’ve ID’d 

the woman inside as Louise Mondelo, the same 

woman identified last weekend in that car that ran 

off Backbone Mountain during the storm. Neither 

body’s in good shape and nothing positive can be 

made until we get DNA, but we need to figure this 

one out.” 

The smell of human decay assaults your nose as you 

go inside. Overturned chairs and tables announce 

the struggle that took place here. The smaller body, 

dressed in a blouse and jeans, lays near the phone 

that now dangles from its line. The larger corpse 

is dressed in a man’s polo shirt and slacks and lays 

in a corner to the left of a door; blood covers the 

walls and floor around him. Another investigator is 

already collecting maggots from the corpses to help 

establish a time of death. As you process the scene, 

you find flesh scraped on the stone of the fireplace, 

and you collect blood and skin from a piece 

of bloody firewood laying near the woman’s body. 

The wounds on her head seem consistent with the 

firewood, but nothing is certain with her in this 

state of decay. Outside of the cabin, you notice a set 

of tire tracks deeply rutted in the mud and grass. 

None of the investigators had driven near that area, 

so you take plaster molds and pictures to preserve 

evidence. 


The Evidence 

Evidence was collected from the remains of the victims 

of the car accident: 

• Adult Woman 

• Female Child 

• Male Child 

Additional evidence was also collected from the victims 

at the fishing cabin, as well as blood evidence 

found at the scene: 

• Adult Male 

• Adult Woman 

• Blood on Firewood 

• Skin Scrapings on Fireplace 

A DNA sample was collected from John Wayne 

Gretzky, a suspect in the cabin murders. Additional 

known samples were collected from the Mondelo 

home. In all, known samples include: 

• Lyle Mondelo 

• Louise Mondelo 

• Wally Mondelo 

• Jan Mondelo 

• John Wayne Gretzky 

22 


The People Involved 

The Mondelos 

Louise Ann Mondelo is the 42 year old mother and 

wife of the Mondelo family. She is also one of the 

owners of Tumbling Water Land Development Company. 

Friends say that Louise was in an unhappy 

marriage and had recently filed for divorce. 

Lyle Christopher Mondelo is the 44 year old husband 

and father of the Mondelo family. He is a 

part owner of Tumbling Water Land Development 

Company and, along with his wife, a partner in L&L 

Mondelo. 

Wally Christopher Mondelo, is the oldest child of 

Lyle & Louise, at 14 years old. During the time that 

Wally was conceived, Lyle suspected Louise of cheating 

on him. Jan Marie Mondelo is 11 years old. 

Other People 

John Wayne Gretzky is 43 years old. He is a friend 

and business partner of the Mondelo’s in the Tumbling 

Water Land Development Company. Rumors 

have it that John Wayne and Louise had a brief affair 

when Lyle and Louise first moved to Highland 

Park. He is known around town to be a greedy businessman, 

and has been suspected of shady deals in 

the past. 

An unknown woman of similar height and build has 

been identified as Louise Mondelo. Although her 

identity is uncertain, this other woman was found 

either driving the Mondelo family car with two 

children preliminarily identified as Wally and Jan, 

or in a remote fishing cabin with a man who has 

been preliminarily identified as Louise’s husband 

Lyle Mondelo. 


Pre-Lab Questions 

DNA Background 

1. What is the key difference between PCR and RFLP? 

2. Why does this difference make PCR better suited for forensics? 

3. What causes DNA to move during electrophoresis? 

4. How is the DNA sorted after electrophoresis? 

5. What is a Ladder? 

Procedure 

6. How much DNA goes into each lane? 

7. What always goes in the first lane of the gel? 

8. Why is it important to write down the loading order of the samples in the wells? 

9. Which direction does DNA travel? Should the wells be on the end near the black or red electrode? 


Lab Procedure 

Preparing the Gel (45 min) 

1. 

2. 

3. 

4. 

5. 

6. 

7. 

8. 

Remove the cap from the bottle of agarose gel 

and microwave in 15-30 second intervals until 

the agarose has completely melted. Watch that 

it does not over-boil in the bottle. Let the agarose 

cool to at least 60°C before continuing. 

Shake the bottle of BlueVis Gel Stain well. 

Transfer 4 mL of BlueVis Gel Stain into the bottle 

with the agarose. Recap and swirl to incorporate 

the stain thoroughly. 

Position the gel box on a level surface and insert 

the gel tray, ensuring that all sides are 

tightly sealed. 

Place the comb at one end of the gel tray. 

Slowly pour the agarose into the gel tray until 

the central cavity and the crevices between 

each tooth in the comb are filled. 

Wait until the gel has become solid and slightly 

opaque (approximately 20—30 minutes). 

Remove and turn the gel tray so that the wells 

are near the black (-) electrode and the rest of 

the gel points to the red (+) electrode. 

Pipetting Order 

Gel A 

Ladder 

Lyle Mondelo 

Louise Mondelo 

Wally Mondelo 

Jan Mondelo 

John Wayne Gretzky 

Preparing the Samples 

Gel B 

Ladder 

Adult Woman (Car) 

Female Child (Car) 

Male Child (Car) 

Adult Woman (Cabin) 

Adult Male (Cabin) 

Firewood (Cabin) 

Fireplace (Cabin) 

The samples will be divided over two gels, A and B, 

to keep the knowns separate from the unknowns/ 

evidence. Both Gels A and B will be run in triplicate; 

that is, three groups will run a copy of gel A 

and three groups will run a copy of gel B. 

Gel A will contain samples that are known; DNA 

for these samples is labeled in Blue. Gel B contains 

unknown samples from the cabin and car accident; 

these samples are labeled in Red. The ladder has 

five lengths of DNA at 150, 300, 500, 650, and 1000 

base pairs. It is labeled in Black and will be run in 

the first lane of both gels. 

1. 

2. 

Collect a set of samples for the gel you will be 

running and set them before you. 

Order your samples according to their pipetting 

order. 

9. 

Fill the gel box with enough 1x BlueVis buffer 

solution provided by your teacher to completely 

cover the gel, making sure that the wells are 

filled. 

10. Carefully remove the comb. 

Loading the Gel 

1. Place a new tip on the pipettor. This should be 

done before each sample. 

2. Ensure pipettor is set to 10 μL. 

3. Tap the sample tube against the palm of your 

hand to settle the contents at the bottom. 

4. Open the sample tube. 

5. Press down on the pipette plunger until you 

reach the first stop. The plunger can continue 


Lab Procedure 

6. 

7. 

8. 

9. 

to go down, but you should notice a distinct 

change in resistance. 

Place the tip of the pipette into the liquid in the 

tube, but do not touch the bottom. Release the 

plunger to obtain the contents of the tube. 

Carefully hover the tip into the appropriate 

well in the gel, being careful not to touch the 

gel itself. 

Press the plunger down slowly to dispense the 

sample into the well. Do not touch the gel itself. 

Remove the pipette from the well before 

releasing the plunger. 

Discard the used tip. 

10. Record the location of the sample on your Data 

Collection sheet. 

11. Moving from left to right, repeat these steps 

for each sample, using a different well and tip 

each time. 

Running the Gel (45 min) 

1. 

2. 

3. 

4. 

5. 

28 

Place the lid down on the top of the gel box. 

Check to see that the black electrode matches 

up with the black lead and red with red. Plug 

the leads into the power supply, also matching 

the colors. 

Before turning on the power supply, make sure 

it reads “Volts” and the setting is “Low.” 

Once all lab stations have been checked, turn 

on the power supply and twist the knob until 

the display reads the voltage that your instructor 

suggests (usually between 60 and 100 volts) 

You should see the orange dye move out of the 

wells within a few minutes. If it has not moved, 

check that your power supply is plugged in, is 

turned on, and is set to the correct voltage. If it 

is, and there is still no movement after several 

minutes, alert your instructor. 

As the gel runs, you will be able to see the DNA 

migrate through the gel. It will begin as a faint 

6. 

7. 

8. 

blue streak near the wells. 

After the gel has run for a time, the blue bands 

should have separated, and grown darker. The 

blue color will intensify as the DNA stains 

while the gel runs. The DNA will never become 

strongly contrasted against the gel, but should 

be clearly visible. 

Run the gel until the orange color in the loading 

dye has migrated about 75% of the way 

through the gel. 

Record the positions of the DNA on the gel on 

your Data Collection sheet promptly. 


Data Collection and Calculations 

Allele 

Name 

vr20 

vr15 

vr10 

vr06 

vr03 

The above schematics should be labeled during the 

lab. Draw the bands seen on your gel on that schematic. 

The ladder has been filled in for you to use as 

a guide. 

Find a group that ran the other gel; copy their 

labels and bands onto this schematic. 

Determining Match Probability 

Not every allele occurs in the general population 

with the same frequency, but, through research, 

these frequencies have been determined. The probability 

that a random person has a given genotype 

can be determined by multiplying the frequencies 

for those alleles together. If the person is heterozygous, 

the combined frequency must be further multiplied 

by 2, because there are two ways to inherit 

those alleles. 

1. For each of the unknown samples, determine the 

probability of a random person having those alleles 

given the frequencies below. 

off 

vr03 vr06 vr10 vr15 vr20 ladder 

0.22 0.31 0.27 0.14 0.05 0.01 

Sample 

Car Wreck 

Adult Woman 

Car Wreck 

Female Child 

Car Wreck 

Male Child 

Cabin 

Adult Male 

Cabin 

Adult Female 

Cabin 

Fireplace 

frequency 

1 

frequency 

2 

Combined 

frequency 


Post-Lab Questions 

Short Answer 

1. Which woman could be Louise Mondelo? 

2. Could the driver of the car be the mother of the two children in the car? 

3. Of the known samples, who could have been Jan’s parents? Wally’s? 

4. Of the known samples, whose DNA could be on the firewood? 

5. Of the known samples, whose flesh could have been left on the fireplace? 

6. Is it easier to prove that two DNA samples match or do not match? 

7. Is it easier to prove guilt or innocence using DNA? 

8. Given that there are only 8,300 people in the county where Highland Park is, how many people in that 

county would you expect to find with the same genotype as each of the unknowns? 

9. Assuming an STR with 5 alleles (ignoring the very rare ‘off ladder’ alleles), how many genotypes exist? 

10. Using the information from #9 above, and assuming that every STR has 5 alleles, on average, how many 

STRs are needed to expect to find only one person of a given genotype in a population of 300 million? 

30 


Mock Trial 

Using this Kit in the Mock Trial 

The DNA evidence analyzed in this lab was only 

from a single STR. It cannot prove conclusively who 

was where or who did what. If the gels were run 

successfully, you should have the following information: 

• The victims in the cabin share the same alleles 

as Louise and Lyle Mondelo. 

• An unknown woman was found dead with victims 

who have the same alleles as Wally and 

Jan Mondelo in the Mondelo family car. 

• DNA from two persons was found on the piece 

of firewood used as a cudgel in the cabin. Of 

the samples tested, the two persons could have 

been any two of Louise, Jan, and the woman in 

the car. 

• An unknown person was present in the cabin 

and left scraped flesh on the stone of the fireplace. 

• Lyle Mondelo is not Wally’s father. Of the samples 

tested, Wally’s father might be either John 

Wayne Gretzky or the unknown male victim at 

the cabin, though approximately 22% of people 

share that allele with Wally. 

If the DNA Typing kit is the only kit done in the 

Mystery of Lyle and Louise suite, a Mock Trial is unlikely 

to be useful since the prosecution does not 

have enough evidence to support a case. Instead, 

use the results as an exercise in the identification of 

human remains. However, if other modules were 

done, a mock trial can help students take all of the 

evidence presented in the background story and 

available from other modules into account, not just 

DNA. Information on running a mock trial follows. 

Before the trial 

If you wish to involve more social studies, have students 

read through the procedures for trial of criminal 

cases and the simplified rules of evidence. Also 

conduct lessons designed to familiarize students 

with the court system and judicial procedure 

Brainstorming 

Using the story and module evidence, list the facts 

of the case on the board. 

Determine, as a class, who should be charged for 

each murder. 

Put students into brainstorming groups. Give all 

of the groups five to ten minutes to come up with 

ideas for each of the following: 

1. 

2. 

3. 

Identify how each fact can support the prosecution’s 

case. 

Identify how each fact can support the defense. 

Identify critical weaknesses in the reliability of 

each fact. 

Review the brainstorming results as a class, and 

have students string facts together to make logical 

assumptions about the case. 

Student Roles 

Have students split up into roles. 

A few of students may need to act as the Court, filling 

the roles of Judge, Bailiff, and Clerk. The Judge 

must research court proceedings and make determinations 

of law; the instructor may wish to take 

this role themselves. The Bailiff is responsible for 

swearing in witnesses and keeping order in the 

court. The Clerk is responsible for recording the 

trial proceedings. You may wish to omit these 

roles, or have these students work with the prosecution 

or defense during the planning stages. If 

you have a large class, some students may also play 

the role of jury. They must attend to the trial proceedings 

and also review the evidence and written 


Mock Trial 

documents prepared by the defense and prosecution 

to come to a conclusion about the case. They 

must then either meet outside of class and come to 

a unanimous decision, or each write a short paper 

justifying their own decision. 

At least one student should act as the forensic scientist 

who processed all of the evidence; if you 

used multiple modules, several students should fill 

this role. This student must be very familiar with 

the laboratory procedures used to process the evidence 

and should also be aware of the ways the evidence 

can be mishandled and the precautions taken 

against evidence contamination and faulty methods, 

as these are likely to come up in court. 

The remainder of students should split, approximately 

evenly, into the prosecution and defense. 

The student filling the role of the accused should 

work with the defense. Each side should assign 

their members as either lawyers or witnesses called. 

The lawyers are responsible for building their case 

and developing the questions to ask their witnesses, 

and also to identify key witnesses called by the 

other side to exploit during cross examination. 

Each side should also identify critical weaknesses 

in their own case and prepare counter-arguments 

for them. Finally, since there are always surprises, 

each side should prepare strategies to deal with the 

unexpected. 

The prosecution must provide a reasonable series of 

events that are consistent with the facts of the case, 

a motive for the events that occurred, and prove beyond 

a reasonable doubt that the accused is guilty. 

The defense may present their own accounting of 

the facts, or undermine the prosecution’s case by 

showing that the prosecution’s witnesses are unreliable, 

that the prosecution’s version of the events 

make no sense or is seriously inconsistent, or by introducing 

reasonable doubt into the prosecution’s 

case. 

when dealing with forensic evidence, and each side 

may wish to call their own, or use the other side’s 

expert. The students playing the role of expert witness 

must become very familiar with that field and 

be able to field questions about the accuracy and 

limitations of the techniques. 

Preparation 

To make sure that students will be ready to argue 

your case, have the prosecution and defense answering 

these questions: 

1. 

2. 

3. 

4. 

5. 

What are the facts of the case? 

Why did these things happen? 

Who was involved? 

Will you have enough evidence necessary to 

participate in the courtroom? 

What is key to you proving your point? 

Have the witnesses answer the following: 

1. 

2. 

3. 

To what are you testifying? 

What are the most important parts of your testimony 

to the prosecution? The defense? 

What holes are in your testimony? If you are 

an expert witness, what are the limitations of 

the evidence presented that is relevant to your 

field? 

Unlike a real trial, witnesses may help the lawyers 

build their case; their primary duty, however, 

should be to become intimately familiar with their 

testimony. Expert witnesses are especially useful 

32

Link to our lab as a pdf - College of Science - Marshall University

Create successful ePaper yourself

Delete template?

Save as template?