Week 7 DNA Electrophoresis Lab

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Tacoma Community College *

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Biology

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May 23, 2024

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Exploring Forensic DNA Analysis: PCR and gel electrophoresis Simona Smith This laboratory assignment will provide opportunities to learn about PCR and gel electrophoresis, and how both technologies are used in forensic science. A fictionalized murder case is included in this lab assignment, and you will analyze the DNA results and help solve the forensics case! Background The Polymerase Chain Reaction (PCR) is essentially a DNA copying mechanism that amplifies specific lengths of DNA (a gene, or part of a chromosome) exponentially! Imagine you have one single piece of DNA in a sample. If you copy it once, you have two copies; copy again, four copies; another copying “cycle,” eight; etc… Imagine doing 30 copying cycles- you would have more than 1 billion copies! In a type of PCR application called End Point PCR, the goal is to make as many copies of the target sequence as possible. You can see the “end point” as billions of copies with your naked eyes as “bands” of DNA on an agarose gel! Gel electrophoresis separates molecules, such as DNA, by exposing the DNA molecules to an electrical current within an agarose gel. An agarose gel is a semi-solid, gelatin-like “slab” which is composed of agarose (polysaccharide found in the cell wall of some types of seaweed) and a salt buffer. Molecules with a net charge can be separated in an electrical field. DNA fragments have an overall negative charge. When exposed to an electrical current, DNA fragments will be attracted to the positive electrode. The DNA fragments will migrate towards the positive electrode (cathode) through the agarose gel. The agarose gel, essentially forms a matrix or a network of pores which causes the separation of DNA molecules based on size (molecular weight). Smaller DNA fragments will migrate faster through the agarose gel and larger DNA fragments will migrate more slowly through the agarose gel. Resources for this Lab Thermo Fisher PCR education https://www.youtube.com/watch?v=Nl6eLez3CNI Agarose Gel Electrophoresis: BioNetwork https://www.youtube.com/watch?v=Swrr_EW8z9Y Lab Assignment Directions Purpose : Write the purpose of this laboratory exercise. Remember to restate the purpose of this lab assignment in your own words Key Terms : Please define the following terms. Remember to restate the terms in your own words  Electrophoresis  Agarose  PCR  Polymorphic  DNA fingerprinting 1

Post Lab Use the resource videos describing PCR and gel electrophoresis to help answer questions 1-4 1. What is the main function of the enzyme Taq DNA polymerase? Taq DNA Polymerase is a thermostable DNA polymerase extracted from the thermophilic bacteria Thermus aquaticus. Its main function is in the polymerase chain reaction technique, where it automates the repetitive step of amplifying specific DNA sequences. 2. Often PCR is described as amplifying DNA. Please explain the meaning of this statement. PCR is polymerase chain reaction. It is a chain of three reactions, which occur one after the other and lead to the amplification of gene of interest. The first step in PCR is denaturation. It occurs at a temperature of around 90 degrees Celsius. In this the double stranded DNA is broken by breaking the hydrogen bonds present between complementary base pairs. It results in the formation of two molecules of single stranded DNA. The second step in PCR is annealing. It is the step in which the primer binds to the 3' end of single stranded DNA. We use two types of primers, Forward primer, and reverse primer. The sequence of primers is complementary to the sequence of genes which we want to amplify. This step occurs at 50 to 60 degrees Celsius. The third step of polymerase chain reaction is elongation. It is the step in which taq polymerase enzyme catalyzes the addition of deoxyribonucleotides at the free hydroxyl group located at the 3’ end. Tag polymerase is a heat stable enzyme, and it is used because PCR is carried out at higher temperatures. This enzyme is isolated from a prokaryote Thermus aquaticus which lives in hot springs. This step occurs at around 70 degrees Celsius. 3. State the reason for using a buffer during gel electrophoresis The buffer in gel electrophoresis is used to provide ions that carry a current and to maintain the pH at a relatively constant value. The buffer transmits the charge through the gel, which separates the molecules by their charge and size. 4. Please explain how DNA migrates through agarose gels. Which 2 factors influence the migration of DNA through agarose gels? Is there a correlation between size of DNA fragments (molecular weight) and migration through an agarose gel. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight. Use the following Case summary and gel electrophoresis results to answer questions 5-7 Summary of murder case A woman has been brutally stabbed to death outside of her home. Two suspects have been arrested. Suspect 1: The woman’s ex-husband, whom the deceased woman claimed had been stalking her in the two months prior to her death 2

Suspect 2: An acquaintance of the women’s ex-husband who had been living in the ex-husband’s house for approximately 6 months and who could not provide an alibi for the time of the murder. DNA was isolated from the following:  Crime Scene: One blood spot found near the victim’s body. (B1)  Crime Scene: One blood spot taken from a glove found near the crime scene. (B2)  Victim (V)  Suspect 1 (S1)  Suspect 2 (S2) Forensic Lab work: The crime lab used PCR analysis to amplify a polymorphic region of chromosome 1. When amplified by PCR, polymorphic DNA regions should show unique patterns of DNA fragments when analyzed by gel electrophoresis. Therefore, different individuals should have unique patterns of DNA fragments (DNA fingerprinting). Figure 1 shows the gel electrophoresis analysis of PCR amplified fragments of the crime scene, suspect and victim DNA. 3

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Source of murder case and image: The Case It Molecular Biology Program http://www.caseitproject.org/ 5. What conclusions can you draw from these results? Which suspect seems to be involved? Just looking at the graph it looks like both subjects where part but subject 1 match with B1 which is one blood spot found near the victim’s body. 6. Do you think this data is sufficient to convict someone? I believe so but I feel like there is a easier way. 7. Name some challenges you experienced interpreting the results. Just reading what subject did what cause its kind of confusing. 4 DNA fragment S2 S1 BS2 BS1 V Well Figure 1 shows the results of DNA analysis of 5 DNA samples isolated from blood samples from 2 suspects (S1 and S2), the victim (V) and 2 blood spots from the crime scene (BS1 and BS2). DNA samples were amplified by PCR and analyzed by gel electrophoresis

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