The Mystery of Lyle and Louise Identity Crisis

GlossaryAgarose: 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 exist as pairs, with one coming from each parent.Comb: A piece of plastic, shaped like a comb, that generates loading wells in agarose gels.DNA: Deoxyribonucleic acid (DNA), a doublestranded, helical nucleic acid molecule that is thecarrier of genetic information.Electrophoresis: When an agarose gel is placedin buffer, DNA samples are placed in wells, and an electrical current is passed through the gel to pull the DNA through the gel. Smaller molecules pass through easily and move away from the wells the fastest, 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. 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.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 piece of plastic which fits onto a pippet. The tips are changed to prevent samples from cross-contamination.

Well: Small rectangular hole in an agarose gel left by comb. Each DNA sample is loaded in a well.

Forensic DNA TechnologyBackgroundEach individual, with the exception of identical twins, has a unique array of genetic informationwithin their deoxyribonucleic acid (DNA). This information is identical in each nucleated cell inthe 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, and that each nucleotide contained a sugar, a phosphate, and one of four different bases. Watson and Crick also knew that the base Adenine (A) and the base Thymine (T) were present in equal amounts, while the base Cytosine (C) and the baseGuanine (G) were present in equal amounts. In addition, Watson and Crick had an X-ray picture of the crystalline structure of DNA by another scientist, Rosalyn Franklin, which showed that 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, and this model is still used today. A negatively charged phosphate/sugar backbone formed the legs of the ladder, pairs of nucleotides, one on eitherside of the ladder, faced the center of the helix and linked via hydrogen bonds to form the ladder’s rungs. T links only with A, and C links only G. Because of this paring, each strand is complimentary to the other and contains the information necessary to build the other strand by serving as a template.

Genes are produced by DNA and determine the characteristics of each individual. Each person has two complete sets of DNA divided into 23 pairs of chromosomes, one from their mother and the other from their father. Each parent may give us the same version, or allele, of a gene (homozygous), or we may 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 traits we possess. It is for this reason that people often exhibit a mixture of their parent’s physicaltraits. Within the entire human population, however, there is less than 0.1% difference within our genetic makeups as 99.9% of your DNA sequence is identical to all of the other people in theworld. The fraction of a percent that varies, however, is enough to produce all of the geneticvariation exhibited between individuals. Not all DNA base combinations 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. These mutations changes are then transfered to that individual’s 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 forensic use of DNA in a 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. Mu14The Mystery of Lyle and Louise echnologyIngredients 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 proteins 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 buildingblocks as DNA. dNTPs (deoxynucleotide triphosphates) is a collective term for any, orall, 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 copying pieces of the 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 iscrucial to the PCR technique, as it assembles raw components into the desired PCR productsunder the higher temperatures needed for the reaction. Three Stages of PCR: PCR is conducted in commercially available instruments, called thermal cyclers, that heat and cool the 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 double strandedDNA ladder are broken, allowing the two strands to separate.

2. Annealing: Temperatures between 50°C to 65°C. The primers bind to the DNA outsidethe targeted region (the region to be amplified) by forming hydrogen bonds with the specific complementary sequences flanking the target region, thus defining the sequence to be copied.

3. Extension (Elongation): Temperature of approximately 72°C. Taq polymerase is activatedand incorporates the free dNTPs into the area between the primers.

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. The concept of PCR resembles 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 be visualized using gel electrophoresis in cases where, prior to this process, it was undetectable.The Mystery of Lyle and Louise 15 Technologystations in DNA, however, may create new cut sites or change the 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 these varying length fragments are compared to fragment size patterns generated from other DNA samples. Unfortunately, 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 a small amount of DNA limiting the usefulness of RFLP analysis.The ability of forensic analysts to copy small amounts of DNA into larger quantities is criticalwhen faced with crime scenes with little biological evidence – a cash register that has only beentouched by a suspect, for example. It is also extremely important because it is 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),vare compared.

The STRs used in DNA typing are located on different chromosomes and follow the genetic 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 other alleles received. Additionally, STRs may be used for paternity testing because half of the offspring’s alleles are inherited from one parent and the other half from the other parent. The figure at the bottom of the page 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 in the figure predicts the possibleallelic combinations for their children. The father is capable of contributing either the 3- or the 6-repeat allele to his offspring, while the mother may contribute either the 2- or the 3-repeat allele. Therefore, as the square indicates, they could potentially 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 works based upon the principle 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 upon 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, made from a component extracted from seaweed which is dissolved in a heated solution, form microscopic pores when the solution cools that act as a molecular sieve through which DNA molecules pass. Possible genotypes of offspring given the genotypes of the mother and father.

orensic DNA TechnologyTo illustrate this concept, the separation matrix may be pictured 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 navigating through the plants than larger fish. Both eventually get through, but the small fish 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 separated DNA fragments cease their migration. Their position in the gel is a direct relation to their size, and these fragments may bestained and their sizes compared to one another.

The figure at the top of the page is a schematic of a stained gel of each of the four possible genotypes of children from the mother and father in the figure on the previous page. Note that 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 as there is twice as much DNA in that area. In electrophoresis, the solution used to create anagarose gel contains a salt dissolved in water, commonly either Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE), that promotes electrical current and stabilizes the gel’s pH. This same solution is also used to submerge the gel, providing an electrically and chemically uniform medium for the electrophoresis process. As the phosphate backbone of DNA is negatively charged in neutral to basic solutions, the solution is pH buffered to prevent acidificationthat would cause the DNA to reverse direction and move toward the negative pole. Following separation by electrophoresis, DNA fragments may be visualized using several methods thatdirectly bind to the DNA molecule. The most common method in an educational setting is to stain with methylene blue. After electrophoresis, the gel is soaked in a stain solution allowing the stain to adhere to the DNA molecules, however, the stain is also absorbed into the pores of the gel. Because of this, the entire gel is blue until allowed to soak in clear water, which dilutes the stain absorbed into the gel in a process called de-staining. 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 utilized. 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 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. As each of these variant forms is called an allele, Gel visualization of genotypes.

Forensic DNA TechnologyThese 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 present on the ladder are common alleles, and 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 locimake up an individual’s genetic profile. Genetic profiles are created for both unknown samplesfrom a crime scene and those that are obtained directly from a person who may be a suspect. Those collected from people, whether a suspect, victim, or any other individual, are referred to as “knowns” and are used to compare known profiles against unknown evidence profiles. An individual is excluded as a suspect when their profile does not match the questioned sample’s profile exactly, and they are included only when the fragment sizes are identicalat every STR locus in the DNA.

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, as 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 illustrated a step forward in this rapidly evolving discipline, however, as technology and laboratory technique grow together further innovations in the science of determining unique identity are assured.

Following the adult female in the vehicle crash on Backbone Mountain and the female homicide victim in the cabin both being identified as Louise Mondelo, investigators collected DNA evidence from both crime scenes in attempt to properly identify the victims.

Samples were collected from all three victims of the vehicle crash, an adult female, a female child, and a male child. Additionally, DNA samples were also obtained from the victims at the fishing cabin, an adult male and an adult female, and from blood evidence found on firewood and skin scrapings on the fireplace at the scene. A DNA sample was also collected from John Wayne Gretzky, a person of interest in the cabin murders, and additional known samples of LyleMondelo, Louise Mondelo, Wally Mondelo, and Jan Mondelo were collected from the Mondelo home.

PERSONS ON INTEREST

PRE LAB QUESTIONS

DNA Background1. 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?

Procedure6. 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?

AlleNamevr20vr15vr10vr06vr03

The above schematics should have been 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.

AlleNamevr20vr15vr10vr06vr03

Copy the labels and bands from a group that ran the other gel onto this schematic.

Determinating Match ProbabilityNot 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 a person is heterozygous, the combined frequency must be further multiplied by 2, because there are two ways to inherit those alleles: allele 1 from the mother and allele 2 from the father or vice versa.1. For each of the unknown samples, determine the probability of a random person having those alleles given the frequencies below:vr03 vr06 vr10 vr15 vr20 Off Ladder0.22 0.31 0.27 0.14 0.05 0.01

SampleFrequency1Frequency2

Combined Frequency 1 Frequency 2 Combined FrequencyCar WreckAdult WomanCar WreckFemale ChildCar WreckMale ChildCabinAdult MaleCabinAdult FemaleCabinFireplace

POST LAB QUESTIONShort Answer

1. Which woman was 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 located, how many peoplein 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?

MOCK TRAIL

Using this Kit in the Mock Trial

The DNA evidence analyzed in this lab was only from a single STR and cannot prove conclusively that a specific person was present or indicate the actions of those present. If everything in the lab was performed correctly you should have obtainedthe 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 possess the same alleles as Wally andJan Mondelo in the Mondelo family car.• DNA from two persons was present 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 inthe 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 samplestested, Wally’s father might be either JohnWayne Gretzky or the unknown male victim at the cabin, though approximately 22 % of peopleshare that allele with Wally.

If the DNA Typing kit is the only kit done in the Mystery of Lyle and Louise, a mock trial is unlikely to be useful as the prosecution has insufficient evidence to support a case. Instead, use the results as an exercise in the identification of human remains.

If other exercises were performed, a mock trial can help students take all of the evidence presented in the investigation and available from other kits into account and provide a more interesting and thorough trial. Information on running a mock trial follows.

Before the trialIf a more thorough social studies activity is desired, students may be instructed to read throughthe procedures for trial of criminal cases and the simplified rules of evidence. Additionally, lessons designed to familiarize students with the court system and judicial procedure may prove beneficial.

BrainstormingUsing the story and module evidence, list the facts of the case on the board. Determine, as a class, who should be charged for each crime.

Put students into brainstorming groups. Give all groups five to ten minutes to develop hypotheses for each of the following:1. Identify how each fact may support the case presented by the prosecution.2. Identify how each fact may support the case presented by the defense.3. Identify critical weaknesses in the reliability of each fact.Review the brainstorming results as a class and instruct students to connect various facts and evidence to make logical assumptions about the case.

Student RolesAllow students to select, or assign, various roles relative to the characters. Additional students may serve as the court, filling the roles of judge, bailiff, and clerk. The judge must research court proceedings and make determinations of law, therefore the instructor may wish to take this role themselves. The bailiff is responsible for swearing in witnesses and keeping order in thecourt. 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. With large classes, students may also play the role of jury. TrialJurors must attend to the trial proceedings and also review the evidence and written 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 an expert witness (the forensic scientist who processed analyzed the evidence presented); if multiple laboratory modules were utilized, 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 defenseteams. 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, developing the questions to ask their witnesses, and for identifying 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 these weaknesses. As there are always surprises during trial, 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 inconsistent, or by introducing reasonable doubt into the prosecution’s case.

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 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.

PreparationTo ensure that students will be ready to argue their case, the prosecution and defense should answer the following questions:1. What are the facts of the case?2. Why did these things happen?3. Who was involved?4. Does sufficient evidence exist to participate inthe courtroom?5. What is key to you proving your point?

Additionally, witnesses should answer the following:1. To what are you testifying?2. What are the most important parts of your testimony to the prosecution? The defense?3. What weaknesses are present in your testimony?If you are an expert witness, what are the limitations of the evidence presented that is relevant to your field?