Directions: Please Return!

Size: px
Start display at page:

Download "Directions: Please Return!"

Transcription

1 Directions: Please Return! Electrophoresis Analysis: restriction enzyme cleavage of DNA Lab AP bio lab 9 (adapted from ) Introduction: Imagine you walk into your AP bio classroom and it looks like a riot scene. Chairs and tables overturned, broken pieces of glass scattered across the floor and several wet red drops. To add to the chaos, it turns out that a person from the class, who was seen around the classroom earlier, has gone missing. What next? What should be done? First, the scene would need to be protected from any disturbances or contamination. Then, criminal investigators would come to analyze the scene and collect evidence. One of the most vital pieces of evidence would be DNA samples that are found on the scene. DNA is unique to every individual and can be used like a fingerprint for identification. Once DNA samples are collected, whether is it is for a criminal investigation or a medical tests for genetic conditions, it can be analyzed using a type of restriction fragment analysis. This includes gel electrophoresis, short fandom repeat (STR) analysis, or restriction fragment length polymorphism (RFLP). Thanks to modern technology, this process is almost completely automated using DNA analysis machines and software. However, this lab takes a look at the basic principles of DNA fragment separation using restriction enzymes and gel electrophoresis. While this lab includes only certain parts of the DNA analysis process, the full I. Isolating DNA: First, DNA must be isolated from a cell. This is easily done by dissolving the phospholipid cell membrane with mild chemical detergents and using the slightly negative charge of the DNA to clump it together and precipitate it out of the cell mixture. Then, if a specific area of DNA is going to be studied, that area must be isolated. This is done with restriction enzymes. II. Copying DNA After isolating DNA or finding a DNA sample for analysis, the DNA needs to be replicated so there are ample amounts of the DNA to work with. Each DNA sample can be multiplied using the polymerase chain reaction (PCR), a process that heads the DNA to break the hydrogen bonds holding the double helix together and then uses the polymerase enzyme to copy the DNA. The process is easily reapeated to create large amounts of DNA. III. Cleaving DNA: The ability to cut and paste, or cleave and ligate, a functional piece of DNA-predictably and precisely is what enables biotechnologists to analyze or recombine DNA-molecules to make recombinant DNA. The first step in DNA-splicing is to locate a specific gene of interest on a chromosome. A restriction-enzyme is then used to cut out the targeted gene from the rest of the chromosome.- Restriction enzymes are proteins that cut DNA at specific sites. Restriction-enzymes, also known as restriction endonucleases, recognize specific sequences-of DNA base pairs and cut, or chemically separate, DNA at that specific-arrangement of base pairs. They were first identified in and isolated from bacteria-that use them as a natural defense mechanism to cut up the invading DNA ofbacteriophages viruses that infect bacteria. Any foreign DNA encountering a-restriction enzyme will be digested, or cut into many fragments, and rendered-ineffective. These enzymes in bacteria make up the first biological immune system.-there are thousands of restriction enzymes, and each is named after the bacterium-from which it is isolated. For example: EcoRI = The first restriction enzyme isolated from Escherichia coli bacteria HindIII = The third restriction enzyme isolated from Haemophilus influenzae bacteria PstI = The first restriction enzyme isolated from Providencia stuartii bacteria Each restriction enzyme recognizes a specific nucleotide sequence in the DNA,-called a restriction site, and cuts the DNA molecule at only that specific sequence.-many restriction enzymes leave a short length of unpaired bases, called a sticky -end, at the DNA site where they cut, whereas other restriction 1

2 enzymes make a cut-across both strands creating double-stranded DNA fragments with blunt ends. In-general, restriction sites are palindromic, meaning the sequence of bases reads the-same forwards as it does backwards on the opposite DNA strand. The specific restriction enzyme will cut all its palindromes, no matter what species the DNA comes from. For example, the image is a list of enzymes and the sites where they cut: IV. Electrophoretic Analysis of Restriction Fragments A restriction enzyme acts like molecular scissors, making cuts at the specific-sequence of base pairs that it recognizes. The three-dimensional structure or-shape of a restriction enzyme allows it to fit perfectly in the groove formed by the-two strands of a DNA molecule. When attached to the DNA, the enzyme slides-along the double helix until it recognizes a specific sequence of base pairs which-signals the enzyme to stop sliding. The enzyme then chemically separates, or cuts,-the DNA molecule at that site called a restriction site. If a specific restriction site occurs in more than one location on a DNA-molecule, a restriction enzyme will make a cut at each of those sites, resulting in-multiple fragments of DNA. Therefore, if a given piece of linear DNA is cut with a-restriction enzyme whose specific recognition sequence is found at five different-locations on the DNA molecule, the result will be six fragments of different lengths.- The length of each fragment will depend upon the location of restriction sites on-the DNA molecule. A DNA fragment that has been cut with restriction enzymes can be separated-using a process known as agarose gel electrophoresis. The term electrophoresis-means to carry with electricity. Agarose gel electrophoresis separates DNA-fragments by size. DNA fragments are loaded into an agarose gel slab, which isplaced into a chamber filled with a conductive buffer solution. A direct current is-passed between wire electrodes at each end of the chamber. Since DNA-fragments are negatively charged, they will be drawn toward the positive pole-(anode) when placed in an electric field. The matrix of the agarose gel acts as a- molecular sieve through which smaller DNA fragments can move more easily than-larger ones. Therefore, the rate at which a DNA fragment migrates through the gel-is inversely proportional to its size in base pairs. Over a period of time, smaller-dna fragments will travel farther than larger ones. Fragments of the same size-stay together and migrate in single bands of DNA. These bands will be seen in the-gel after the DNA is stained. An analogous situation is one where all the desks and chairs in the classroom-have been randomly pushed together. An individual student can wind his/her way-through the maze quickly and with little difficulty, whereas a string of four students-would require more time and have difficulty working their way through the maze. Making DNA Visible DNA is colorless so DNA fragments in the gel cannot be seen during-electrophoresis. A loading dye containing two blue dyes is added to the DNA-solution. The loading dye does not stain the DNA itself but makes it easier to load-the gels and monitor the progress of the DNA electrophoresis. The dye fronts-migrate toward the positive end of the gel, just like the DNA fragments. The faster -dye comigrates with DNA fragments of approximately 500 bp, while the slower -dye comigrates with DNA fragments approximately 5 kb in size. Staining the DNA-pinpoints its location on the gel. When the gel is immersed in Fast Blast DNA stain,-the stain molecules attach to the DNA trapped in the agarose gel. When the bands-are visible, your students can compare the DNA restriction patterns of the different-samples of DNA. 2

3 This Lab: You will be provided with lambda DNA and three different restriction enzymes. Lambda DNA is the genomic DNA of a bacterial virus, or bacteriophage (phage), which attacks bacteria by inserting its nucleic acid into the host bacterial cell. Lambda is a phage that replicates rapidly inside host cells until the cells burst and release more phages to carry out the same infection process in other bacterial host cells. Bacteriophage lambda is harmless to man and other eukaryotic organisms, and therefore makes an excellent source of DNA for experimental study. The DNA restriction analysis that you are about to perform is fundamental to a variety of genetic engineering techniques, including gene splicing, DNA sequencing, gene localization, forensic DNA matching, or DNA fingerprinting. * answer all pre-lab questions in lab report Materials: 20 ul vial of DNA fragments prepared using restriction enzymes rack for holding samples micro pipette and tips permanent marker gel electrophoresis chamber power supply staining tray semi-log graph paper ruler 0.8% agarose solution (or prepaired gel) 1x TAE (tris-acetate-edta) buffer methylene blue stain Practice Using Micropipets Practice using a micropipete with water or food coloring. Practice pipetting drops of water onto a paper towel with hand drawn rectangle to represent wells in the gel. 1. Look at the micropipet to determine the volume range. 2. Twist the dial on the micropipet to set the desired volume to 5 ul (05.0). 3. Affix a tip to the pipettor by firmly jamming the end of the pipettor into a tip in the rack. 4. Press the micropipet plunger to the first (soft) stop. 5. Insert the pipet tip into the solution to be transferred. 6. Slowly release the plunger to retrieve the liquid. 7. Insert the pipet tip into the desired tube. 8. Press the plunger past the first stop to the second (hard) stop to transfer the-liquid. Make sure to keep the plunger pressed when lifting the pipet tip out of-the tube. 9. Eject the tip from the pipettor after using it. Always do this after loading each sample, otherwise the samples will be contaminated. v Be careful that you do NOT push to the second stop when drawing in liquid, only when expelling liquid. Lesson 1: Restriction Digestion (this may be done by the instructor) Laboratory Procedure: The DNA you will be provided with has been extracted from a bacteriophage a bacterium-invading virus. The virus is known as lambda and is often written as λ. You will be working with three different restriction enzymes, also called endonucleases. These are referred to as PstI, EcoRI, and HindIII. 1. Obtain micro test tubes that contain each enzyme solution, lambda DNA, and restriction buffer from the common station. Keep all the stock solutions on ice. 2. Label four micro test tubes L, P, E, and H and place them in the foam microtest tube holder. L = Uncut lambda DNA P = PstI restriction digest of lambda DNA E = EcoRI restriction digest of lambda DNA H = HindIII restriction digest of lambda DNA 3

4 3. You will set up your digests in micro test tubes. To each tube, add 4 µ l of uncut lambda DNA, 5 µ l of restriction buffer and 1 µ l of enzyme. Add only one kind of enzyme to a tube. Do not add enzyme into the tube labeled L. Use the chart to keep track: Tube Lambda DNA Restriction PstI EcoRI HindIII Buffer P 4µl 5µl 1µl E 4µl 5µl µl H 4µl 5µl µl L 4µl 5µl Tightly cap each tube. In order to mix all reagents, hold the top of a micro test tube between the index finger and thumb of one hand and flick the bottom of the tube with the index finger of the other hand. Gently tap the bottom of the tub on the table to collect the liquid. If you are using a centrifuge, place the four tubes from your tube into the centrifuge, being sure that the tubes are in a balanced arrangement in the rotor. Have your teacher check before spinning the tubes. Pulse-spin the tubes (hold the button for a few seconds). 5. Place the sample tubes in a 37 C water bath for approximately 30 minutes or let them incubate at room temperature overnight. Restriction enzymes work-best at 37 C since they were isolated from bacteria that live inside warm-blooded-animals. After the incubation, place the samples in the refrigerator (4 C) untilthe next laboratory period, or proceed directly to step 2 of Lesson 2 if instructed-by your teacher. While waiting: cast your agarose gel: 1. Seal the ends of the gel tray securely with strips of standard duct tape. Cut the duct tape about 4 long. Press the tape firmly to the edges of the gel tray to form a fluid-tight seal. Make sure at least ¼ of tape is sealed under the tray. This is important so the gel doesn t leak. 2. Level the gel tray on a table or work bench and place the comb at the negative end. 3. Prepare the desired concentration and amount of agarose in 1 x TBE or 1 x TAE electrophoresis buffer. a. To Make 6 or 7 gels: add 2.4g of agarose to 300ml of 1X buffer b. To Make 1 gel: add.5g agarose to 60mL of 1x buffer 4. Using the Magnetic Hot Plate, hot plate, or microwave, heat the solution to boiling while stirring. Boil the solution until all of the small translucent agarose particles are dissolved. With the small flask still in place, set aside to cool to 60 degrees C before pouring gels. 5. Pour enough agarose to cover the gel comb teeth or to a depth of cm. Use 6. Do not move or handle the gel tray until the gel has solidified. 7. Allow the gel to solidify at room temperature for at least 20 minutes it will appear cloudy (or opaque) when it is cooled off. 8. Place on a tray and put the tray in the refrigerator if you need to store overnight. *answer question for Lesson 1 in lab report 4

5 Lesson 2: Agarose Gel Electrophoresis Part 1. Prepare Your Samples for Electrophoresis Consideration 1. How Can Fragments of DNA Be Separated From One Another? DNA is colorless so DNA fragments in the gel can t be seen during electrophoresis. A sample loading buffer containing two blue dyes is added to the DNA solution. The loading dyes do not stain the DNA itself but makes it easier to load the gels and monitor the progress of the DNA electrophoresis. The dye fronts migrate toward the positive end of the gel, just like the DNA fragments. The faster dye comigrates with DNA fragments of approximately 500 bp, while the slower dye comigrates with DNA fragments approximately 5 kb, or 5,000 bp, in size. 1. Following incubation, obtain your four micro test tubes L, P, E, and H and place them in the foam micro test tube holder at your laboratory desk. Place on ice if possible L = Uncut lambda DNA (yellow tube) P = PstI restriction digest of lambda DNA (violet tube) E = EcoRI restriction digest of lambda DNA (green tube) H = HindIII restriction digest of lambda DNA (orange tube) 2. Set the digital micropipet to 2.0 µ l and transfer this amount of sample loading dye to each of the tubes marked L, P, E, and H in the foam tube holder. Referense the instruction for using a micropipette. Use afresh tip with each sample to avoid contamination. 3. The DNA samples and the sample loading dye must be thoroughly mixed in each tube before placing the samples in the gel wells for electrophoresis. This is easily accomplished by holding the top of a microtube between the index finger and thumb of one hand and flicking the bottom of the tube gently with the index finger of the other hand. Collect the liquid to the bottom of the tube by tapping it gently on your laboratory bench. Ask the instructor to use the centrifuge if the DNA is liquids are not mixing. 4. Locate the DNA marker (M) from your teacher. Optional: Heat all samples at 65 C for 5 minutes and then place the samples on ice this results in better separation of the DNA bands. Part 2. Set Up Your Gel Electrophoresis Chamber 5. Obtain your agarose gel. 6. Place the casting tray, with the solidified gel in it, onto the central platform in the gel box. The wells should be at the negative (cathode) end of the box where the black electrical lead is connected. 7. Pour about 275 ml of electrophoresis TAE buffer into the electrophoresis chamber. Pour enough buffer into the box until it just covers the wells of the gel by 1 2 mm. 8. Very carefully remove the comb from the gel by gently pulling it straight up. Part 3. Load your Samples and Run them by Electrophoresis 9. Pipet 5-10 µl from each tube ( M, L, P, E, and H) into separate wells in the gel chamber. Use a fresh tip for each tube. Gels are read from left to right. To keep things straight, the first sample is typically loaded in the well at the upper left-hand corner of the gel. If you notice that a well in the gel has been compromised (the wall between it has been broken), skip that well and use the next one. If you have pleny of lanes, it can be good to skip a lane between samples: Example: 5

6 optional: At this point, if available, you can also add extra unknown DNA samples or unknown dye samples that simulated unknown DNA to the extra lanes on the other side of your gel. This will be provided by your teacher. Make sure to skip as many lanes as you can between your DNA and these sample, starting on the right side of your gel instead of the left. Lane Sample: suspect 1 suspect 2 crime scene 10. Slide the cover of the chamber into place, and connect electrical leads to the power supply, anode to anode (red to red) and cathode to cathode (black to black). Make sure both electrical leads are attached to the same channel of the power supply. 11. Plug the gel chamber into the power supply, black to black and red to red. Turn the power on and set the voltage to 100V and press the run button. 12. Electrophorese at 100 V for minutes (ask instructor as needed). If it is working, small bubble can be seen coming up through the chamber. Shortly after the current is applied, the loading dye can be seen moving through the gel toward the positive side of the gel chamber. 13. When electrophoresis is complete, turn off the power supply, disconnect the leads from the inputs, and remove the top of gel chamber. 14. This is a good time to take a picture of your gel so far. If you added in the optional DNA samples in the form of a dye, you will need to analyze these now, as this dye will disappear when you go so stain your gel. 15. Remove the casting tray from gel chamber. The gel is very slippery. Hold the tray level. 16. Pour the excess buffer back into the labeled container for reuse. Part 4: Staining DNA with Fast Blast DNA Stain There are two protocols for using Fast Blast DNA stain in the classroom. Use option 1 for quick staining of gels to visualize DNA bands in minutes, and option 2 for overnight staining. Depending on the amount of time available, your teacher will decide which protocol to use. Two student teams will stain the gels per staining tray (you may want to notch gel corners for identification). If needed, mark staining trays with initials and class period on masking tape before beginning this activity. WARNING Although Fast Blast DNA stain is nontoxic and noncarcinogenic, avoid skin contact to keep hands from becoming stained blue or wear latex or vinyl gloves while handling the stain or stained gels. Lab coats or other protective clothing can be worn to avoid staining clothes. Protocol 1: Quick Staining of Agarose Gels in 100x Fast Blast DNA Stain This protocol allows quick visualization of DNA bands in agarose gels within 15 minutes. For quick staining, Fast Blast DNA stain (500x) should be diluted to a 100x concentration. We recommend using 120 ml of 100x Fast Blast to stain two 7 x 7 cm or 7 x 10 cm agarose gels in individual staining trays provided in Bio-Rad's education kits. If alternative staining trays are used, add a sufficient volume of staining solution to completely submerge the gels. Following electrophoresis, agarose gels must be removed from their gel trays before being placed in the staining solution. This is easily accomplished by holding the base of the gel tray in one hand and gently pushing out the gel with the thumb of the other hand. Because the gel is fragile, special attention must be given when handling it. We highly recommend using a large spatula or other supportive surface to transfer the gel from one container to another. Destaining requires the use of at least one large-volume container, capable of holding at least 500 ml, at each student workstation. Each student team may utilize separate washing containers for each wash step, or simply use a single container that is emptied after each wash and refilled for the next wash. 1. Mark the staining trays with your initials and class period on masking tape. You will stain 2 gels per 6

7 tray. 2. Stain gels: Remove each gel from the gel tray by carefully sliding it into the staining tray. Pour approximately 100 ml of 100x stain into the staining tray. If necessary, add more100x stain to completely submerge the gels. Stain the gels for 2 3 minutes, but not for more than 3 minutes. Using a funnel, pour the 100x stain into a storage bottle and save it for future use. The stain can be reused at least 7 times. 3. Rinse gels: Transfer the gels into a large container containing ml of clean, warm (40 55 C) tap water. Gently shake the gel in the water for ~10 seconds to rinse. 4. Wash gels: Transfer the gel into a large container with ml of clean, warm tap water. Gently rock or shake the gel on a rocking platform for 5 minutes. If no rocking platform is available, move the gels gently in the water once every minute. 5. Wash gels: Perform a second wash as in step Pour off the water and examine the stained gels for expected DNA bands. The bands may appear fuzzy immediately after the second wash, but will begin to develop into sharper bands within 5 15 minutes after the second wash. This is due to Fast Blast stain molecules migrating into the gel and binding more tightly to the DNA. To obtain maximum contrast, additional washes in warm water may be necessary. Destain to the desired level, but do not wash the gel in water overnight. If you cannot complete the destaining in the allocated time, you may transfer the gel to 1x Fast Blast stain for overnight staining. See Protocol 2. Protocol 2: Overnight Staining of Agarose Gels in 1x Fast Blast DNA Stain For overnight staining, Fast Blast DNA stain (500x) should be diluted to a 1x concentration. We recommend using 120 ml of 1x Fast Blast to stain two 7 x 7 cm or 7 x 10 cm agarose gels in individual staining trays provided in Bio-Rad's education kits. If alternative staining trays are used, add a sufficient volume of staining solution to completely submerge the gels. Following DNA electrophoresis, agarose gels must be removed from their gel trays before being placed in the staining solution. This is easily accomplished by holding the base of the gel tray in one hand and gently pushing out the gel with the thumb of the other hand. Because the gel is fragile, special attention must be given when handling it. 1. Mark the staining tray with your initials and class period. You will stain 2 gels per tray. 2. Stain gels (overnight)* Pour 1x stain into a gel staining tray. Remove the gel from the gel tray and carefully slide it into the staining tray containing the stain. If necessary, add more 1x staining solution to completely submerge the gels. Place the staining tray on a rocking platform and agitate overnight. If no rocking platform is available, agitate the gels staining tray a few times during the staining period. You should begin to see DNA bands after 2 hours, but at least 8 hours of staining is recommended for complete visibility of stained bands. * It is crucial that you shake gels gently and intermittently while performing the overnight staining in 1x Fast Blast stain since smaller fragments tend to diffuse without shaking. 3. No destaining is required after staining with 1x Fast Blast. The gels can be analyzed immediately after staining. *answer question for Lesson 2 in lab report Lesson 3: Analysis of Results 1. Place your gel on a light background and record your results by making a diagram, taking and printing a picture, or making a tracing with a clear sheet of plastic. (put in lab report) 2. You can rry the agarose gel as a permanent record of the experiment: a. If a smaller gel is desired, you can first trim away any unloaded lanes with a knife or razor blade. Cut your gel from top to bottom to remove the lanes that you did not load samples into, leaving only lanes used. b. Place the gel directly upon the hydrophilic size of a piece of gel support film. (or wax paper) 7

8 (Water will form beads on the hydrophobic side of a piece of gel support film.) Center the gel on the film on a paper towel and let the gel dry in a well-ventilated area, making sure to avoid direct exposure to light. As the gel dries it will bond to the film but will not shrink. If left undisturbed on the support film, the gel will dry completely at room temperature after 2 3 days. The result will be a flat, transparent, and durable record for the experiment. Note: Avoid extended exposure of dried gels to direct light to prevent bandfading. However, DNA bands will reappear if the dried gels are stored in thedark for 2 3 weeks after fading. Calculating the Size or Restriction Fragment Length Polymorphisms Mathematical formulas have been developed for describing the relationship between the molecular weight of a DNA fragment and its mobility (i.e., how far it migrates in the gel). In general, DNA fragments, like the ones in your evidence samples, migrate at rates inversely proportional to the log10 of their molecular weights. For simplicity s sake, base pair length (bp) is substituted for molecular weight when determining the size of DNA fragments. Thus, the size in base pair length of a DNA fragment can be calculated using the distance the fragment travels through the gel. To calculate the base pair length, a DNA standard, composed of DNA fragments of known base pair length, is run on the same gel as the unknown fragments and is then used to create a standard curve. The standard curve, in this case a straight line, is created by graphing the distance each fragment traveled through the gel versus the log10 of its base pair length. 1. As explained above, base pair (bp) length is substituted for molecular weight. Note that in plotting the standard curve, calculating the log10 of the base pair length of each fragment is unnecessary because the base pair size is plotted on the logarithmic axis of semi-log paper. Examine your stained gel on a light colored surface that helps visualize the bands. 2. Measure the distance (in mm) that each fragment migrated from the origin (the well). Hint: For consistency, measure from the front end of each well to the front edge of each band, i.e., the edge farthest from the well. Enter the measured distances into Table 1 for Distance Traveled in mm. If there are some bands missing or one DNA sample that did not turn out, that is ok. (If your gel did not turn out well enough at all, use the ideal gel image provided) 3. Plot the data from the DNA sample cut with HindIII on semi log paper. To do this, use distance traveled as the (arithmetic) x-axis and the base pair (bp) length as the (logarithmic) y-axis. Don t forget a title for your graph. 4. Connect the data point with a best-fit line by connecting as may of the points as you can with a straight line. (However, you may need ignore the point plotted for the 27,491bp/23,130 doublet. When using 0.8% agarose gel, these fragments appear as one). Congratulations! Your best-fit line is the standard curve. 5. Now use the standard curve to calculate the approximate sizes of the EcoRI and BamHI fragments. Use a ruler to help guide you. Fill in the rest of Data Table 1 *answer question for Lesson 3 in lab report and do your conclusion 8

9 Names: Electrophoresis Analysis: restriction enzyme cleavage of DNA Lab Report Prelab questions: 1. Summarize the purpose of this lab: Per: row: 2. What process is used to make enough DNA copies of the DNA to use? 3. What are restriction enzymes? 4. What processes are used to analyze DNA fragments? Lesson 1: Restriction Digestion: answer the following based on lesson 1 1. In which micro-tube do you expect no changes to occur that is, no DNA fragments produced. (what is the control)? What is missing in that tube that leads you to that decision? 2. Is there any visible change to the DNA after adding restriction enzymes? 3. If the DNA molecule has two restriction sites, A and B, for a specific restriction enzyme (image below), how many fragments would be produced if the DNA is cut by that enzyme? Lesson 2 Agarose Gel Electrophoresis 4. It what direction do DNA fragments move, in reference to the charged poles in the chamber? 5. Which fragments move the farthest, small or large? Why? 6. A piece of DNA is cut into four fragments as shown in the diagram. A solution containing the four fragments is placed in a well in an agarose gel. Using the information given above, draw (to the right) how you think the fragments might be separated. Label each fragment with its corresponding letter. 7. What is the significant of the Fast Blast or methylene blue stain? 9

10 8. In the rectangle below, draw in the number of gel lanes you have and label what was in each lane by writing the name down the lane. This will help you identify what is what later in the procedure. Lane 1 has been placed for you M: DNA marker + (the bottom of the gel, has been cut off to save space) 10. At the end of electrophoresis, before staining your DNA, what does the gel look like? If you added the optional DNA dyes, can you make a match between the crime scene and a suspect? Lesson 3: Analysis of Results 11. Attach the picture, plastic sheet tracing, or do your drawing of your gel here, after it has been stained: 10

11 Data Table 1: Title: 11

12 12. What evidence do you have that each enzyme cuts the DNA at different locations? 13. Now look at the diagram of the agarose gel (below). It shows two lanes. A lane is the column below a well. The right lane contains a banding pattern from four fragments of known length (6,000, 5,000, 3,000, and 1,000 bp). a. Which lane contains the molecular weight standards? How do you know? b. Label each band in the right lane witits base-pair size. c. Compare the two lanes of bands. Estimate the size of the fragments in the left lane. Upper band: Lower band: Conclusion: (attach paper if needed) 1. Summaries the process of DNA analysis. (you may use bullet points) 2. What are some possible sources of error in the electrophoresis procedure? How can you minimize any potential sources of error? 3. What if the overall significance of using DNA analysis techniques? How are they applied? 4. There are important social and ethical implications of DNA analysis. Already, DNA testing can reveal the presence of markers of certain genetic diseases, such as Huntington s Suppose a DNA test that predicted your chances of getting a disease, such as cancer, were available. You take the test for cancer, and the results say you have a two in three chance of getting cancer sometime in the next 20 years. Who should have access to your genetic profile and this information? Your doctor? Health insurance companies? College admission officers? Employers? Would you want to know this information? Who owns your DNA and its information? 12