Materials Included In Kit

Additional Materials Required

Prelab Preparation

Part 2. DNA Isolation Lab Activity

1. Put the ethyl alcohol in a lab freezer or on ice for at least 30 minutes before the lab.
2. For each group, prepare three dropper bottles:
   1. Add 3 mL of 8% sodium chloride solution to one of the dropper bottles.
   2. Add 3 mL EDTA solution to one of the dropper bottles.
   3. Add 3 mL SDS solution to the last dropper bottle.
   4. Label each bottle.
3. Pour 10 mL of drinking water into 16 clean, 30-mL cups.

Part 3. Modeling Forensic DNA Analysis
1. Make 8 copies of each DNA Sequence Template sheet.
2. Cut out the primers and the dNTPs and group them with the corresponding DNA Sequence Template sheets.
3. Sort pop beads into containers for each group according to the following chart.

Safety Precautions

Ethyl alcohol is flammable and a dangerous fire risk; keep from flame and sources of ignition. Only place ethyl alcohol in a certified laboratory freezer. Sodium dodecyl sulfate solution causes skin and serious eye irritation. Provide students new clean drinking cups. Never allow students to drink or eat from an apparatus in the lab. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The resulting mixtures can be rinsed down the drain according to Flinn Suggested Disposal Method #26b.

Lab Hints

• Enough materials are provided in this kit for 8 groups of 4. Two groups will combine for Part 3. Extra pop beads and connectors are included.
• This module can reasonably be completed in four, 50-minute class periods. Complete the POGIL™ activity on day one, the demonstration on day two and modeling of DNA forensics on days 3 and 4.
• Emphasize that in actual DNA forensics analysis, the length of the primers and the number of repeats is much longer, generally between 100 and 350 bp.

Teacher Tips

• This module can be utilized during a genetic unit to teach DNA structure and biochemical interactions.
• Students should have background information on types of bonds, including hydrogen bonding and the roll of polarity in intermolecular forces.
• The following student laboratory kits can be used to further explore DNA structure and separation techniques: PCR-Based DNA Fingerprinting Kit (Catalog No. FB1892) and PCR Simulation—Student Activity Kit (Catalog No. FB1749)

Answers to Prelab Questions

1. Which parts of the genome are used in STR forensic analysis?

   Noncoding regions of four repeating nucleotides. Some are introns from known genes while most are not associated with genes.

2. STR markers vary between individuals in what unique way?

   STR markers vary in the number of times the four repeating nucleotides occur.

3. Explain why there is so much variety in the DNA of STR markers compared to the coding regions of DNA.

   Noncoding regions are not subject to natural selection because they have no influence on the traits of the person. Since there is no selective pressure, there are many different alleles for each of these markers.

Sample Data

Part 2. DNA Isolation Lab Activity 
Complete the following table showing how each step in the isolation process separates DNA from other substances.

DNA Isolation Process

Answers to Questions

Part 2. DNA Isolation Lab Activity

1. Assign each of the steps from the table above to a different member of your group. Draw a model of the step showing how the chemical added interacts with the cells and/or DNA in the sample.

   Answers will vary but should show how the DNA changes location or shape due to the addition of the chemical.

2. Share the models among your group and make changes based on feedback within your group. Record any changes to your models.

   Answers will vary.

3. Compare the two different samples of DNA collected in your group. Note the quantity in each. Explain why there may be differences.

   Answers will vary. Acceptable answers include differences in the technique used during separation or differences in the amount of cheek cells collected.

Part 3. Modeling Forensic DNA Analysis 

1. Explain how heating and cooling facilitates bonding and releasing during PCR.

   Bonding can only occur at specific temperatures and these temperatures are unique to each type of molecule. Heating and cooling allows hydrogen bonds between molecules to form and release.

2. Describe four differences between the simulation of DNA forensics and the actual process.

   Answer will vary. Acceptable responses include:

   • STR markers are much longer in real DNA samples.
   • There is more variation between individuals than shown in the simulation.
   • There are twenty CODIS STR markers and at least eight must be present in a sample. The simulation only looked at four.
   • Each individual has two alleles for each marker. The simulation looked at one allele for each sample.
   • PCR doubles the number of copies of the STR markers more than twenty times, resulting in over one billion copies.
   • Visualization occurs automatically using a genetic analyzer.
3. Why must a sample contain at least 8 STR markers to be used for forensic analysis?

   The probability of two individuals having the exact same genotype for eight or more STR markers is statistically impossible except in cases of identical twins. If less than 8 STR markers are present in the sample, that probability increases and the possibility exists that two samples may falsely match.

Final Analysis

1. Using the claims, evidence, reasoning model, make a claim about who may have committed the murder according to the DNA evidence. Defend the claim with specific evidence and use scientific information to back it up.

   Answers may vary.
   
   Claim: Suspect 2 is the only person who may have committed the murder.
   
   Evidence: The Visualization of Results shows that all four markers from the crime scene DNA sample match Suspect 2. Suspect 1 only matches on two of the markers so can be excluded as a suspect. The victim’s DNA does not match the crime scene DNA, therefore the crime scene DNA does not belong to the victim.
   
   Reasoning: STR analysis relies on exact matches between the crime scene and the suspect. Without an exact match, the suspect can be excluded from consideration. In this simulation, only four markers are used, therefore another four markers must be found and analyzed to make a conclusive match that the DNA at the crime scene is the DNA of Suspect 2. Once that is confirmed, other evidence must point to the Suspect to show that the DNA left at the crime scene was connected to the murder. For example, if Suspect 2 was the victim’s significant other, their DNA would be expected at the crime scene but if Suspect 2 was a stranger and was claiming they had never been at the crime scene, the DNA evidence would show that Suspect 2 had been at the crime scene.

Teacher Handouts

11409_Teacher.pdf

References

Butler, John and Dennis Reeder. “FBI CODIS Core STR Loci.” Short Tandem Repeat DNA Internet Database. Accessed July 24, 2017. http://strbase.nist.gov/fbicore.htm

Butler, John. “Short tandem repeat typing technologies used in human identity testing.” BioTechniques 43: Sii-Sv (October 2007) doi 10.2144/000112582

“DNA Structure and Replication.” POGIL™ Activities for High School Biology. Trout, L., Editor; Flinn Scientific: Batavia, IL (2012).

Introduction

Understanding the structure of DNA was one of the first steps to unlocking the biochemistry behind heritability. Chargaff used paper chromatography in his discovery that adenine and thymine were found in equal amounts; and cytosine and guanine were found in equal amounts. Shortly after, Rosalind Franklin’s X-ray diffraction of DNA led to Watson and Crick’s groundbreaking discovery that the primary structure of DNA is a double helix with evenly spaced intervals between the bases. The first DNA fingerprints used restriction enzymes to cut DNA into fragments that were uniquely sized for each person. Currently, PCR technology is used to tag and amplify fragments of DNA called STR markers.

Concepts

• DNA structure
• Gel electrophoresis
• Primers
• DNA isolation
• PCR
• Short tandem repeat

Background

Part 1. Establishing Background Knowledge

The POGIL™ activity is designed to be completed in class using the POGIL™ teaching method. This includes students working in groups with assigned roles to construct their own learning using modeling. For more information, visit www.pogil.org.

Part 2. DNA Isolation Lab Activity

Isolating DNA is the first step to being able to analyze a sample. The DNA must be separated from the other types of molecules in the sample using a combination of chemicals. These chemicals organize the types of molecules by dissolving and precipitating them.

The process of DNA extraction, regardless of the tissue used, involves the same key steps:

1. The sample is shaken or blended with a salt solution. The sodium ions in the salt attach to the phosphate ends of the DNA, protecting them from dissolving and pushing them closer together. This makes it easier to precipitate the DNA out of solution.
2. A detergent is added that breaks down and emulsifies the fat and proteins that make up the cell membrane. This allows the DNA to escape from the nucleus, and the sample is shaken or blended again.
3. The solution is treated (with heat or chemically) to break down any DNase enzymes present, which could digest the long DNA strands into smaller pieces making spooling more difficult. Sometimes additional enzymes are added to help digest the fat and protein molecules.
4. DNA is soluble in water and insoluble in ethanol. The addition of ethanol causes the DNA to precipitate and come out of solution. The DNA precipitates at the water/alcohol interface allowing it to be “spooled” onto a spooling device.

Part 3. Modeling Forensic DNA Analysis

This activity teaches the process of DNA forensics analysis. DNA analysis involves several steps and may vary in technique depending on available technology. The current method used by the FBI is called STR analysis. In this activity, you will simulate the steps in STR analysis using pop beads to find out who left their DNA at a crime scene.

Within the long strands of DNA observed in the previous activity are coding portions and noncoding portions of DNA. Coding portions get transcribed into RNA that moves out of the nucleus where it is translated to make proteins. By altering the coding and noncoding portions, a single gene can make many different proteins. However, there are portions of the genome that are almost never used in the coding of proteins. These are repeating DNA base pair sequences that vary much more than genes. Repeating sequences between two and six base pairs are called short tandem repeats, or STR markers.

The FBI has identified twenty STR markers that are all four base pairs in length and have a great deal of variety between individuals. An additional STR marker on the Y chromosome helps identify the biological sex of the person’s DNA. These twenty-one markers make up the Combined DNA Index System, better known as the CODIS database, from which DNA fingerprinting is performed. When DNA is analyzed, at least eight of the markers must be present in a sample to be used as evidence. Table 1 shows the repeating pattern of nucleotides and location of each marker. Each possible allele is distinguished by the number of repeats contained in the marker. Since STR markers do not code for proteins, there is no selective pressure to narrow down the variation, so many potential alleles can exist for a single marker. This means that if eight or more of the markers are present and match, investigators can be certain that they have a DNA match.

In this modeling simulation, you will carry out the processes shown in Figure 1 on four different strands of DNA associated with a murder. Samples were collected from the crime scene, the victim and two potential suspects. Your team will conduct analysis of the four samples to see if either suspect left their DNA at the crime scene. In order to simplify the simulation, your class will do the first part, PCR, for only four STR markers, each of which is shorter than the actual marker. In reality, the tagged primers are 10 to 30 nucleotides long and the number of repeats may exceed 40. The simulation is based on STR markers D2S441, D8S1179, D10S1248 and TH01. The first three are from noncoding regions of the DNA. TH01 is from the first intron in the human tyrosine hydroxylase gene.

The ingredients necessary for polymerase chain reaction, or PCR, are a DNA sample, primers consisting of short sequences of DNA, DNA nucleotide bases known as dNTPs, Taq polymerase enzyme and an appropriate buffer. When PCR is used for forensics analysis, fluorescent tags are added to the primers so the DNA that binds to the primer sequence can later be identified. PCR has three steps: denaturing, annealing and extending. This process is accomplished by heating and cooling the components to specific temperatures for specific time intervals using a thermocycler. The steps are outlined:

• High temperature denatures the double-stranded DNA sample into single strands.
• The temperature is lowered so that primers can attach via hydrogen bonding to both the top and bottom strands by pairing with complementary base pairs during annealing.
• The last stage, extending, occurs when the temperature is raised, allowing the Taq polymerase enzyme to bind next to the primer and begin synthesizing new DNA using the dNTPs in much the same way that DNA polymerase does during replication. When the temperature lowers, copying stops. The thermocycler will raise and lower the temperature up to thirty times to make over one billion copies of the STR marker. A fluorescent label binds to the primer region of each new copy.

After PCR, the amplified DNA is separated using gel electrophoresis or capillary electrophoresis. Both use electrical current and a matrix to separate the STR markers by size. Remember that the size varies from person to person because each person has a specific number of repeats. The fluorescent labels are specific to each STR marker, so all the markers can be analyzed at the same time by looking for the different colored bands. A genetic analyzer will digitize the results and report the sizes of each STR marker as a separate line on a graph.

Experiment Overview

In this module, you will explore the properties of DNA that allow scientists to perform DNA fingerprinting. First, use the DNA Structure and Replication POGIL™ activity to learn about the structure of DNA, base pairing rules and DNA replication. Then extract and isolate DNA from your own cheek cells. Finally, solve a murder mystery by building models of STR markers and simulating PCR amplification and separation. Analyze how the intermolecular forces of the various molecules allow for the isolation, amplification and separation of DNA.

Materials

Prelab Questions

Part 3. Modeling Forensic DNA Analysis

1. Which parts of the genome are used in STR forensic analysis?
2. STR markers vary between individuals in what unique way?
3. Explain why there is so much variety in the DNA of STR markers compared to the coding regions of DNA.

Safety Precautions

Sodium dodecyl sulfate solution causes skin and serious eye irritation. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Part 1. Establishing Background Knowledge 

In groups, complete the DNA Structure and Replication POGIL™ activity. 

Part 2. DNA Isolation Lab Activity

1. In your group of four, choose two people to donate their DNA sample. Complete the following steps on each sample. Record your observations in the table on the DNA Structure Worksheet.
2. Add 1 mL (20 drops) of the 8% sodium chloride solution to the larger test tube. Set the tube aside in a test tube rack.
3. Pour 10 mL of fresh tap water or bottled water into a clean drinking cup.
4. Put the 10 mL of water in your mouth and “swish” the water around between your cheek and gums on one side of your mouth for at least 30 seconds. Spit the water back into the plastic cup. (The swishing of the water washes cells from inside your cheeks into the water.)
5. Pour several mL of the “cheek cell” water into the test tube containing the salt solution from step 1.
6. Add 1 mL (20 drops) of the 10% SDS solution and 1 mL (20 drops) of the 0.1 M EDTA solution to the “cheek” mixture in the test tube.
7. Stopper the test tube and mix the contents of the tube by gently inverting the test tube several times. Do not shake the test tube. The SDS breaks down the cell membrane from the cheek cells, releasing the DNA into the salt solution. The EDTA solution inactivates the DNA digesting enzymes.
8. Holding the test tube at a slight angle, carefully add 5 mL of ice cold 95% ethyl alcohol down the side of the test tube so that it forms a layer over the “cheek” mixture in the test tube (see Figure 2). Do not mix the water and ethyl alcohol layers.
   {11409_Procedure_Figure_2}
9. Hold the test tube upright for 1–3 minutes and observe what happens at the interface between the ethyl alcohol and the “cheek” solution. Note the development of a cloudy, stringy precipitate at the interface.
10. Add about 1 mL (20 drops) of 95% ethyl alcohol to the smaller, empty test tube.
11. Place a clean inoculating loop in the test tube containing the DNA. Collect the DNA by turning the loop in one direction and thus winding the DNA strands around the loop.
12. Carefully remove the loop and DNA from the solution and transfer it to the smaller test tube containing 1 mL of 95% ethyl alcohol. Observe the DNA floating in the alcohol.
13. Complete the Post-Lab Questions for Part A on the DNA Structure Worksheet.
14. Consult your instructor for appropriate disposal procedures.

Part 3. Modeling Forensic DNA Analysis

Part A. Isolate DNA

1. Combine two groups from the POGIL activity to form an investigative team of eight students. A pair of team members will be responsible for analyzing one set of DNA. Assign responsibility as follows:

   Pair 1: Crime scene DNA
   Pair 2: Victim’s DNA
   Pair 3: Suspect 1 DNA
   Pair 4: Suspect 2 DNA

2. Use the appropriate pop beads and connectors to assemble the assigned DNA marker. Assemble the nucleotides from the 3′ to 5′ strand of DNA first. Then assemble the complementary 5′ to 3′ strand. Follow the sequence shown on the DNA Sequence Template specific to your assigned marker. Refer to Figure 3 to review how to attach the three parts of the nucleotide together.
   {11409_Procedure_Figure_3}

   Use the following color scheme to assemble the DNA strands:

   • Adenine—yellow
   • Cytosine—blue
   • Deoxyribose—white
   • Guanine—green
   • Phosphate—red
   • Thymine—orange
   • Connectors—clear
3. Connect the two strands using the clear connectors, which represent hydrogen bonds. Remember to connect A–T and C–G across the two strands.
4. Make Taq polymerase by connecting 6 black pop beads in a circle.
5. Orient all the molecules so that the 3′ to 5′ is on top and the 5′ to 3′ is on the bottom.

Part B. PCR

1. Gather the following items. This mixture of items represents the components needed for PCR.
   1. The double strand of DNA made in Part A.
   2. Primer with fluorescent labels that corresponds with the STR marker you are working on.
   3. One Taq polymerase molecule.
   4. dNTP strands.
2. The items above have been loaded into the thermocycler and you have set the program. First, the temperature goes up to 95 °C to denature the double-stranded DNA. Denature the DNA sample by breaking the hydrogen bonds between the two strands. Separate the two strands of DNA, keeping the 3′ to 5′ on top and the 5′ to 3′ strand on the bottom
3. The next step is annealing. In this step, the temperature decreases to 50 °C, allowing the primers to attach to both the top and bottom strand. Note: The actual temperature depends on the exact primers being used and must be calculated. Attach primer to the complementary DNA segment on the 3′ to 5′ strand. Remember to pay close attention to the 5′ and 3′ markers on the primers so they line up properly. The primer will have a fluorescent tag attached to it.
4. The third step is extending. The temperature increases to 68 °C, allowing the Taq polymerase to bind to each strand near the primer and new nucleotide bases to attach via hydrogen bonding. Place the Taq polymerase around the first nucleotide in the STR marker. Find the complementary base pair sequence from the dNTP strands. The enzyme Taq polymerase enables the dNTPs to form hydrogen bonds with the existing strand at this temperature. The final copy will have the fluorescent tag and primer still attached.
5. This completes the first round of PCR. In actual PCR, the thermocyclers will continue to raise and lower the temperature as seen in Table 2, with the DNA doubling with each round. Table 2. PCR Timing using Taq polymerase
   {11409_Procedure_Table_2_PCR timing using Taq polymerase}

Part C. Gel Electrophoresis

1. Compete this step with four people in your group that each performed PCR on a different sample of DNA. Double check that you have the information for the crime scene, the victim, suspect 1 and suspect 2.
2. Locate a Paper Gel Electrophoresis Template and record the STR marker that your group is analyzing.
3. For each of the four samples in your group, make a horizontal mark on the paper corresponding to the number nucleotide bases found in the sample including the primer, but not including any bases outside the primer. Use the top strand of DNA to determine this. Use a marker that matches the color of the fluorescent tag. This represents the distance the STR marker travels through the gel. The lower the number of repeats, the further it goes. Note: In actual STR analysis, each sample has two alleles for each marker, one on each of the homologous chromosomes. For simplification, this lab looks at just one of the alleles for each.

Part D. Visualization

1. To quickly analyze results from many different DNA samples, the data from all the available markers at a crime scene are processed into line graphs showing the quantity of DNA found for each possible genotype. This is typically done using a genetic analyzer and accompanying software. See Figure 1 in the Background section for an example of what these results look like.
2. Using the Visualization worksheet, locate the point on each of the four graphs corresponding to the number of nucleotide bases in the primer and STR marker combined. This point should be at the height of the 1 on the y-axis, indicating 1 allele. Use a color corresponding to the color of the fluorescent tag to mark this point on the graph for each DNA sample.
3. Collect data on each of the other three STR markers from people in the other groups. Use a color corresponding to the color of the fluorescent tag for each of these three STR markers to mark the appropriate points on the graph for each DNA sample.
4. At each point, make a peak so that the graphs resemble those in Figure 1, step 4: Visualization. The peaks may be closer together because the variation in the lengths of the STR markers is not as large as they are in actual DNA samples.
5. Complete the Post-Lab Questions on the DNA Structure Worksheet.

Student Worksheet PDF

11409_Student.pdf