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Poppy McDonald
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1 Polymerase Chain Reaction Lab: a Forensic Application INTRODUCTION PCR (polymerase chain reaction) is a technique that scientists use to amplify particular segments of DNA. This process can produce large quantities of the specific DNA sequence from a very small sample of DNA. In theory, only a single template strand is needed to generate millions of new DNA molecules. Applications of this technique include analysis of genetic mutations, identifying missing persons, paternity, infectious agents, and forensic analysis of crime scenes. How can DNA evidence solve crimes? One way scientists can use this technique is to analyze an individual s genotype. Every person's genotype is their own uniquely personal genetic barcode. Crime scenes often contain biological evidence (such as blood, semen, hairs, saliva, bones, or pieces of skin) from which DNA can be extracted. DNA profiling refers to the use of molecular genetic methods to determine the genotype of a DNA sample. If the DNA profile obtained from evidence discovered at the scene of a crime matches the DNA profile of a suspect, this person is potentially guilty; if the two DNA profiles do not match, the individual is excluded from the suspect pool. What kinds of human DNA sequences are used in crime scene investigations? Out of 3 billion base pairs in the human genome, more than 99.5% are identical in all human beings. However, approximately 0.5% of the human DNA sequence does differ. Variations in DNA sequence between individuals are termed "polymorphisms" (many forms). These special polymorphic sequences are used in forensic applications. By universal agreement, DNA sequences used for forensic profiling are "anonymous"; that is, they come from regions (loci) of our chromosomes that do not control any known traits and have no known functions. The DNA sequences used in forensic labs are non-coding regions that contain segments of Short Tandem Repeats or STRs. STRs are very short DNA sequences (2-5 base pairs) that are repeated in direct head-totail fashion. STRs are found surrounding the chromosomal centromere (the structural center of the chromosomes). The example below shows a locus (known as TH01) found on chromosome 11; its specific DNA sequence contains four repeats of [TCAT]. The polymorphisms in STRs are due to the different number of copies of the repeat element that can occur in a population of individuals...c C C T C A T T C A T T C A T T C A T T C A.. The TH01 STR locus has more than 20 different alleles worldwide that differ from each other by the number of [TCAT) repeats. Each person has two of these alleles, one from our mother and one from our father. Figure 1 shows the alleles of two suspects: suspect A has one allele with 5 repeats, and one allele with 3 repeats, giving a DNA profile for the TH01 locus of 5-3; suspect B has one allele with 6 repeats, and one allele with1 0 repeats, giving a DNA profile for the TH01 locus of Suspect A s DNA type for the TH01 locus is (5-3) Suspect B s DNA type for the TH01 locus is (6-10) C C C A A A 5* C C C A A A 6* C C C A A A 3* C C C A A A 10* * Number of (TCAT) repeats Figure 1. Two sample TH01 genotypes. When DNA is amplified by PCR and the fragments analyzed by agarose gel electrophoresis, a genetic profile can be determined. These profiles can be compared to DNA found at a crime scene in order to identify the perpetrator. In this experiment, you will perform PCR analysis on a single locus, the BXP007 locus, using template DNAs obtained from a simulated crime scene and suspects. Following PCR, you will run an agarose gel to separate the PCR products, visualize the PCR products, compare them to a simulated ladder of possible 8-1

alleles for this locus, and assign a genotype for the templates. You will then look to see if any of the suspects' genotype match the crime scene, and see whether you can determine whodunit! 1.

2 alleles for this locus, and assign a genotype for the templates. You will then look to see if any of the suspects' genotype match the crime scene, and see whether you can determine whodunit! 1. What kinds of materials obtained from a crime scene might contain DNA? 2. Why do you need to perform PCR on DNA obtained from a Crime Scene? What might you see if you ran a DNA sample extracted from evidence on a gel before PCR? 3. What is a genotype? 4. What is the difference between an allele and a locus? 5. Why do forensic labs analyze non-coding DNA and not genes? PCR Amplification PCR amplification is DNA replication in a test tube. The portion of the DNA you want to make copies of is called the target sequence. The sample of DNA obtained at a crime scene and the suspect's DNA samples contain the target sequence. PCR reactions occur in 3 steps: 1. Denaturation. Before new DNA synthesis can begin the double stranded DNA template must be unwound and separated into single strands. In cells this is carried out by a family of enzymes. In PCR, heat is used to melt apart - or denature - the double stranded DNA template by raising the temperature to around 94 o C. 2. Annealing. Before a target region of DNA can be amplified, primers must be constructed so that DNA polymerase has available 3 ends to add onto. Primers are carefully designed so that they hybridize to complementary sequences of the template DNA at either end of the sequence to be amplified. In PCR, primers must anneal, or bind, to their respective complementary base pair sequences on the template. The target sequence is determined by where the primers anneal. Annealing is done by lowering the temperature to a point at which binding of primers to template is favored, typically around 55 o C. 3. Extension. Once the primers have annealed, DNA polymerase can bind and replicate, or extend, the target sequence. A special DNA polymerase must be used for PCR because most enzymes would denature during the denaturation step. A DNA polymerase was isolated from the bacterium Thermus aquaticus (Taq), which lives in high-temperature steam vents such as those found in Yellowstone National Park. This Taq polymerase is heat stable and is ideal for PCR reactions. Extension is done at around 72 o C, which is the optimal temperature for Taq polymerase. 8-2

3 These 3 steps - denaturation, annealing, and extension - together make up one PCR cycle. A complete PCR reaction involves many repetitious of a single PCR cycle. In this experiment, your PCR reactions will cycle 35 times. With each PCR cycle, the number of DNA copies doubles. Therefore, after 35 cycles, the DNA of interest has been amplified sufficiently to be visualized using gel electrophoresis. Only a few components are needed for a PCR reaction: DNA template, primers, Taq DNA polymerase, deoxyribonucleotides, and a buffer to maintain ph and salt concentration. In your reaction, all of the components except the DNA template are premixed for you into a Master Mix. It's important to keep the Master Mix cold to keep the enzyme inactive. The DNA templates have been collected from the crime scene and 3 suspects. Your task is to amplify the region of interest (the BXP007 locus, a polymorphic allele) from the DNA samples. Once complete, you will analyze your PCR products using gel electrophoresis to determine the genotypes of the samples at the BXP007 locus and match the crime scene DNA to one of the suspects. 1. List the components you need to perform PCR and state why you need each component. Which of these components are contained in the Master Mix? 2. What steps make up a PCR cycle and what happens at each step? GOALS Understand the processes of DNA amplification and gel electrophoresis. Simulate a crime scene investigation and figure out which suspect committed a crime. PART 1: PCR AMPLIFICATION You are about to conduct real world forensic DNA profiling. As a crime scene investigator, you will use the polymerase chain reaction (PCR) and agarose gel electrophoresis to analyze the DNA samples obtained from a hypothetical crime scene and four suspects. Your job is to figure out which of the four suspects is the perpetrator. Imagine the following scenario: Scene: The Highway Motel, #1 Dark Highway, Nowhere Setting: Room #2131 The motel manager hears loud voices, a woman screams, and a shot rings out. The manager runs to the window in time to see the receding lights of a car leaving in a hurry. The door to room # 2131 hangs open. The manager runs to the open door, to see a man lying face down in a pool of blood. He calls 911. The police arrive, and begin to examine the crime scene. It is an apparent homicide, but with no obvious clues as to who committed the crime. 8-3

4 You are a forensic specialist who is called in to examine the crime scene and collect evidence. Even though it looks like the people involved left no evidence behind, you can use laboratory tests that can tell who was at the crime scene from a single drop of blood or semen or a lone hair. Detectives on the case have identified four suspects and have obtained blood samples from each one. Your task: figure out which suspect is the murderer. MATERIALS Ice bath + Styrofoam cup+ float 4 microcentifuge adaptors (capless tubes) DNA from Crime scene and suspects 1 sharpie 1 1 tube of Master Mix P-20 ul micropipette with tips 4 PCR tubes PROCEDURES 1. Label 4 PCR tubes CS, A, B, and C and include your group name or initials as well. 2. Use a micropipetter to add 10ul of Master Mix to each PCR tube. Add 10ul of the appropriate template DNA (from the provided tubes) to each PCR tube, using a fresh micropipette tip for each DNA template. After adding the DNA template to each tube, mix by gently pipetting up and down. Keep tubes on ice as much as possible. 3. Close the caps on the PCR tubes and place them in their adaptors on ice. You can remove the template DNA tubes to make room for your PCR tubes. 4. When instructed to do so, place your tubes in the thermal cycler rack with those of your classmates. Your instructor will place the rack into the thermocycler and run the PCR reaction before your next class period. It will take about 4 hours. Teacher Signature: PART 2. GEL ELECTROPHORESIS OF PCR PRODUCTS Your PCR amplification is complete but you cannot see the amplified DNA. Gel electrophoresis allows us to sort your PCR products by length and then visualize them using a DNA stain. Since DNA is negatively charged, it can be separated using an electric current. In agarose gel electrophoresis, DNA is placed in solidified agarose, which contains tiny pores. An electric current is applied across the gel. As the current travels through the gel, it carries the negatively charged DNA toward the positive anode. Larger molecule of DNA moves more slowly through the gel so they do not travel as far. Smaller molecules move more quickly and travel farther. In addition to your PCR products, you will also be running a DNA Allele Ladder that represents all of the possible alleles at the BXP007 locus. This is a reference, or marker, that you can compare your PCR reactions to so you can judge their relative sizes and their identities. An example of a possible outcome is shown in Figure 3. The first lane contains the Allele Ladder (AL). The next lane contains the Crime Scene Sample (CS) and the next 3 contain DNA from the suspects. 8-4

Obtain your 4 PCR reactions from the previous lesson, place them into a capless tube adaptor, and place both tubes into a rack. 2.

5 Figure 3 MATERIALS 4 PCR Reactions from previous lab one P-20 micropipette with tips Pre-stained 1.5 % agarose gel Gel tank & Power Supply Running Buffer PROCEDURE 1. Obtain your 4 PCR reactions from the previous lesson, place them into a capless tube adaptor, and place both tubes into a rack. 2. Each group will load 10ul of each of their samples onto the agarose gel according to the table below. Lane Sample Load volume 1 Crime Scene 10 ul 2 Suspect A 10 ul 3 Suspect B 10 ul 4 Suspect C 10 ul 3. Your instructor will start the gel and run it at volts for approximately 15min. RESULTS: 4. Who did it? Explain your reasoning. 8-5

Overview. Background ~30 min. Lab activity ~50 min. DNA profiling Polymerase Chain Reaction (PCR) Gel Electrophoresis PCR

Overview. Background ~30 min. Lab activity ~50 min. DNA profiling Polymerase Chain Reaction (PCR) Gel Electrophoresis PCR Overview Day 1: Tuesday Introduction to DNA profiling How do we use DNA to solve crimes? Background Polymerase Chain Reaction (PCR) Gel Electrophoresis Set up PCR Day 2: Wednesday Make and Run Agarose