This lab also contributes to the attainment of the following elements of the 00UK objective:

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1 General Biology I The Unity of Life Laboratory Introduction to PCR 10% of lab mark (2% of final course mark) modified from: BioRad Biotechnology Explorer Crime Scene Investigator PCR Basics. This lab activity is intended to provide an opportunity for students to: become aquainted with some contemporary biotechnology procedures; apply links between technology and the theories of genetics and population genetics; gain experience analyzing data and drawing simple conclusions from those data. Students will work in teams of three. Objectives: This lab aims to contribute to the development of the following components of the Cégep Champlain St. Lawrence Science Program Graduate Profile: I. Apply The Experimental Method II. Take A Systematic Approach To Problem Solving III. Use The Appropriate Information Technologies IV. Reason Logically V. Communicate Effectively VI. Learn In An Autonomous Manner VII. Work As Members Of A Team VIII. Make Connections Between Science, Technology and Social Progress X. Become Familiar With the Context in Which Scientific Concepts are Discovered and Developed XI. Develop Attitudes Appropriate For Scientific Work XII. Apply What They Have Learned To New Situations This lab also contributes to the attainment of the following elements of the 00UK objective: 1. To recognize the relationships between the structures and functions of certain levels of organization of living beings. 2. To analyze the mechanisms that are responsible for the genetic variation of living beings. This lab also contributes to the development of the following performance criteria of the 00UK objective: Proper use of concepts and terminology Clear description of the principal steps of a biological process. Accurate description of structures and their functions. Description of the correlations between structures and functions. Appropriate use of the laws of genetics and the chromosome theory of heredity. Observance of the experimental method and, where applicable, the experimental procedure. Adherence to safety and environmental protection regulations. Appropriate use of techniques of observation and experimentation. Pre-lab Assignment Due September 30, 2008 Each student is required to provide the answers to the following questions: 1. Examine the photos of the January 15, 2008 crime scene in the biology lab (available on the St. Lawrence server). Which of the available evidence items could be sources of DNA? Justify your choices. (4 points) 2. Compare DNA synthesis as it occurs in a normal, eukaryotic cell with DNA synthesis as it occurs during PCR (polymerase chain reaction). Briefly and clearly state three (3) elements that the two processes have in common as well as two (2) significant differences between the processes. (5 points) 3. What is the difference between an allele and a locus? (1 point) 4. Suspect C s DNA type for the TH01 locus is (5-9). What do the numbers 5 and 9 refer to? (1 point) What inferences can be made about the genotypes of Suspect C s parents? (2 points) 1

2 Background Information: In this lab, you will use the polymerase chain reaction (PCR) and agarose gel electrophoresis to analyze the DNA samples obtained from a crime scene and four suspects. Your job is to identify the perpetrator. In this analysis, a genotype is the particular set of genetic markers, or alleles, in a DNA sample. Every person s genotype is their own uniquely personal genetic barcode In this experiment, you'll be revealing the genetic barcodes of several individuals, and looking for a match. How can DNA evidence solve crimes? DNA profiling refers to the use of molecular genetic methods used to determine the genotype of a DNA sample. This powerful tool is routinely used around the world for investigations of crime scenes, missing persons, mass disasters, human rights violations, and paternity. Crime scenes often contain biological evidence (such as blood, semen, hairs, saliva, bones, pieces of skin) from which DNA can be extracted. If the DNA profile obtained from evidence discovered at the scene of a crime matches the DNA profile of a suspect, this person is included as a potentially guilty person; if the two DNA profiles do not match, the individual is excluded from the suspect pool. PCR is DNA replication gone crazy in a test tube PCR produces large amounts of a specific piece of DNA from trace amounts of starting material (template). The template can be any form of double-stranded DNA. A researcher can take trace amounts of DNA from a drop of blood, a single hair follicle, or a cheek cell and use PCR to generate millions of copies of a desired DNA fragment. In theory, only a single template strand is needed to generate millions of new DNA molecules. Prior to PCR, it would have been impossible to do forensic or genetic studies with this small amount of DNA. The ability to amplify the precise sequence of DNA that a researcher wishes to study or manipulate is the true power of PCR. One of the main reasons PCR is such a powerful tool is its simplicity and specificity. The specificity of PCR is its ability to target and amplify one specific segment of DNA a few hundred base pairs in length out of a complete genome of over 3 billion base pairs. In addition, all that is required for PCR is at least one DNA template strand, DNA polymerase, two DNA primers, and the four nucleotide building block subunits of DNA A, G, T, and C otherwise known as the deoxynucleotide triphosphates of adenine, guanine, thymine, cytosine, and reaction buffer. PCR allows forensic scientists to reveal personal details about an individual's genetic makeup and to determine the most subtle differences in the DNA of individuals - from the tiniest amount of biological material. The fact that millions of exact copies of a particular DNA sequence can be produced easily and quickly using PCR is the basis for modern forensic DNA testing. What kinds of human DNA sequences are used in crime scene investigations? There are ~3 billion basepairs in the human genome greater than 99.5% do not vary between different human beings. However, a small percentage of the human DNA sequence (<0.5%) does differ, and these are the special polymorphic ("many forms") sequences used in forensic applications. By universal agreement, DNA sequences used for forensic profiling are "anonymous"; that is, they come from regions of our chromosomes (also called loci) that do not control any known traits and have no known functions. Loci are basically genetic addresses or locations. A single locus may have different forms or types; these different forms are called alleles. A locus may be biallelic, having only two different forms, or it may be polymorphic, as described above. 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 that are repeated in direct head-to-tail fashion. The example below shows a locus (known as TH01) found on chromosome 11; its specific DNA sequence contains four repeats of [TCAT]. 2

3 Suspect A s DNA type for the TH01 locus is (5 3) Suspect B s DNA type for TH01 locus is (6 10). Figure 1. Two sample TH01 genotypes. For the TH01 STR locus, there are many alternate polymorphic alleles that differ from each other by the number of [TCAT] repeats present in the sequence. Although more than 20 different alleles of TH01 have been discovered in people worldwide, each of us still has only two of these, one inherited from our mother and one inherited from our father. For example as shown in figure 1, 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. How are STR alleles detected? The key to DNA profiling is amplification of the copies present in the small amounts of evidentiary DNA by polymerase chain reaction (PCR). Using primers specific to the DNA sequences on either side of the [TCAT] STR, billions of copies of each of the two original TH01 alleles in any one person's DNA type are synthesized in the reaction. These copies contain the same number of STRs present in the original DNA copies and can be visualized using agarose gel electrophoresis. By comparison with a DNA size standard, or allele ladder, that corresponds to the known sizes of TH01 alleles, the exact sizes of the PCR products from the sample DNAs can be determined and compared. A diagram of the results for TH01 typing of Suspect A and Suspect B is shown in figure 2. In this cartoon example, PCR has been performed on DNA from 2 suspects using primers specific for the TH01 locus. Following gel electrophoresis which separates the PCR products according to their size, the pattern of bands is compared to the Allele Ladder to identify the alleles present in the original samples. Imagine a scenario in which Suspect A and Suspect B are accused of being involved in a love triangle and committing the murder of a third person in the Highway Motel; the person who actually pulled the trigger is unknown. In addition to DNA samples from the crime scene, the forensic specialist will isolate DNA from suspects, victims, and any others present to genotype as controls. Using PCR-based analysis, the samples will be examined at 13 different genetic locations, or loci, using software to interpret the results from the amplification products. In real crime scene analysis, DNA profiling is performed at many loci to improve the power of discrimination of the testing. In simple terms, the power of discrimination is the ability of the profiling to tell the genetic difference between different individuals. The larger the number of loci profiled, the more powerful the ability to discriminate. Fig. 2. Illustration of sample TH01 genotypes following gel electrophoresis. 3

4 Scene: Cégep Champlain-St.Lawrence Setting: Biology Lab, room 343; January 15, 2008; 8:30 am On their first day of class of the winter 2008 session, the students of CSI: St. Lawrence were shocked by a bloody scene in the biology lab: an overturned stool, bloody footprints, blood splattered on the fume hood, countertops and floor, and a bloody baseball bat left unceremoniously in the corner of the room. After cordoning off the apparent crime scene, the students took photos and carefully collected evidence. Interviews with witnesses identified four individuals who had been observed in, or near, the biology lab in the early hours before class, but no one heard or saw anything out of the ordinary. The CSI students proposed a couple of possible scenarios to explain the crime scene but were unable to identify the victim or the perpetrator. Who should be retained for further questioning and who should be eliminated as a suspect? In this experiment, you will perform PCR analysis on a single locus, the BXP007 locus, using DNA extracted from the collected evidence and suspects. Following PCR, you will run an agarose gel to separate the PCR products, visualize the PCR products, compare them to a ladder of possible alleles and assign a genotype to each of the samples. You will then determine which of the suspects genotypes match the DNA found at the crime scene and determine which individuals can be eliminated as suspects. Suspect A: chemistry teacher Suspect C: physical education teacher Suspect B: biology teacher Suspect D: technician 4

5 Polymerase Chain Reaction September 30 Required Materials bucket of crushed ice Master Mix* + primers (MMP) evidence collected from crime scene samples collected from suspects (4) PCR tubes (5) yellow pipet tips P-20 micropipet P-200 micropipet *Note: the Master Mix contains Taq DNA polymerase, deoxynucleotides (A, T, C, G) buffers and salts. Procedure 1. Examine the ice bucket at your work area. In the ice you should find: One yellow tube labeled MMP (master mix + primers) containing a blue liquid Five (5) microtubes containing DNA samples labeled: CS (purple tube) crime scene A (green tube) suspect A B (blue tube) suspect B C (orange tube) suspect C D (pink tube) suspect D 2. Label five (5) PCR tubes with the codes: CS, A, B, C, and D and your group name or initials. 3. Add 20 µl DNA to each appropriately labeled PCR tube. For example, transfer 20 µl of the Crime Scene DNA sample into the PCR tube labeled 'CS'. IMPORTANT: USE A FRESH PIPET TIP FOR EACH DNA SAMPLE. a. Set the P-20 to 20 µl. Add a sterile tip to the shaft of the P-20. Press the tip on firmly with a slight twisting motion to insure a positive, airtight seal. To prevent contamination, avoid touching the tip with your fingers. Close the lid of the pipette-tip box when finished. b. Depress the plunger of the micropipet to the first stop. Holding the micropipet vertically, immerse the disposable tip into the DNA sample to a depth of 1-2 mm. c. Let the pushbutton return SLOWLY to the up position. Never allow it to snap up. Wait 1-2 seconds before removing the tip from the solution. d. To transfer the DNA sample, place the tip against the inside wall of the PCR tube near the bottom and depress the plunger slowly to the first stop. Wait one second, then depress the plunger to the second stop (bottom of stroke) to expel any remaining liquid in the tip. e. Keep plunger fully depressed when withdrawing the micropipet. f. Discard the tip directly into the biological waste bag by depressing the tip ejector button. 4. Use the P-20 micropipet to add 20 µl of the Master Mix + primers to each PCR tube. Mix the contents of the PCR tubes by gently pipetting up and down. IMPORTANT: USE A FRESH PIPET TIP EACH TIME. Once you ve added MMP to a tube, close the cap. The solution in your PCR tubes should be blue. If it s not blue, ask for assistance. 5. Place your capped PCR tubes on ice. 6. When instructed to do so, place your tubes in the thermal cycler. (35 cycles of: 94 C, 30 sec; 52 C, 30 sec; 72 C, 60 sec) 5

6 Electrophoresis of PCR Products October 7 Required Materials 3% agarose gel PCR Samples from previous lab 1X TAE running buffer P-20 micropipet Orange G loading dye (LD; orange liquid) Gel staining tray yellow pipet tips Gel electrophoresis chamber Power supply DNA stain Crime Scene Investigator Allele Ladder (orange liquid) microcentrifuge Procedure Loading and running gels 1. Practice loading an agarose gel with fake samples before loading your PCR products. Do not pierce the bottom of the well with the micropipet tip. Do not overload wells. 2. Place the agarose gel in its tray into the electrophoresis chamber. Slowly add 1X TAE running buffer into one side of the chamber until the buffer is level with the top of the gel. Add buffer to the other side of the chamber until the buffer is level with the top of the gel. Continue to slowly add buffer until the level is approximately 2-3 mm above the gel. 3. If necessary, pulse spin your PCR samples in the centrifuge to position the contents in the bottom of the tube. 4. Set a P-20 micropipet at 10 µl. Use the P-20 to add 10 µl of Orange G loading dye (LD) to each of the PCR samples and mix well by gently pipetting up and down. IMPORTANT: USE A FRESH PIPET TIP EACH TIME. 5. Using the Gel Loading diagram on page 8 as a guide, load 20 µl of the allele ladder and 20 µl of each sample into the appropriate well. 6. Place the cover on the electrophoresis chamber so that the red cord is connected to the red terminal and the black cord connects to the black terminal. 7. Run the gel at 100 V for 30 minutes. Do not let the orange dye front migrate out of the gel. Procedure Staining agarose gels 1. Add 120 ml of 1X Fast Blast DNA stain to a staining container. 2. When electrophoresis is complete, turn off the power and remove the lid from the electrophoresis chamber. Carefully remove the gel in its tray from the electrophoresis chamber. Be careful, the gel is very slippery. Nudge the gel off the gel tray with your thumb and carefully slide it into a container for staining. 3. Let the gels stain for approximately 4 24 hours with gentle shaking for best results. No destaining is required. 4. When staining is complete, the teacher or technician will photograph the stained gel or copy the banding pattern. A photocopy of the image will be provided to each team. 6

7 Analysis of Results October 21 Once the gels have been stained, it is time to determine the alleles present in each sample, and assign DNA profiles (genotypes). For each PCR reaction, compare the bands obtained in each lane to the Allele Ladder. Refer to the figure below for representative results, sizes of the ladder bands, and labeling of the alleles in the ladder. Assign each band in each PCR reaction with an allele assignment according to the band of corresponding size in the allele ladder. The bands in the allele ladder are numbered from top to bottom starting with the largest allele, #15, at the top. Write down the genotype for each of your samples in the chart below. Lane Sample Number of bands Genotype of sample 1 BXP007 Allele Ladder 2 Crime Scene DNA 3 Suspect A DNA 4 Suspect B DNA 5 Suspect C DNA 5 Suspect D DNA 7

8 Loading Plan for two rows of 20 wells L CS A B C D L CS A B C D L CS A B C D team 1 team 2 team 3 L CS A B C D L CS A B C D L CS A B C D team 4 team 5 team 6 8

9 Post-lab Assignment Due November 4, 2008 Each team is required to provide the answers to the following questions: 1. Provide the genotype of each of your samples. 2. Based on the genotype analysis, which suspect(s) can be eliminated and which suspects should be retained? Justify your choice. 3. Suppose that each allele at the BXP007 locus is found at exactly the same frequency in a population. Since there are 8 possible alleles at the BXP007 locus, what is the frequency of any one allele from this locus in this population? 4. Using the assumed allele frequency described in question 3, what is the frequency of the genotype of the DNA found at the crime scene? Show your work. 5. If you had a pool of 13 suspects, and only one suspect had a genotype that matched the BXP007 locus at the crime scene, would the evidence from only the BXP007 profile be convincing enough to convict the suspect? Explain. 9