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Name________________ Section_______________ Spring 2007 DNA Testing Lab Biology 212 Genetics Laboratory Goals of the Lab: To expose students to technologies used for DNA testing and applications for their use in medical diagnostics and forensics. Reference and Background Information: European Initiative for Biotechnology Education at http://www.eibe.info/ Lab Procedure for DNA Testing Simulation 1. A 0.6% (w/v) agarose gel in 1xTBE must be prepared about a half hour ahead of the lab period. Most likely, we will prepare gels the day before lab and store them for use the next day. For the small gels, weigh out 0.3 g agarose, add 50 ml 1xTBE and heat for about 3 min. in the microwave. Add 2 microliters of 1 mg/ml ethidium bromide after the solution has cooled slightly. Pour into a gel tray that has been sealed on the ends with tape. A comb should be placed near one end. Larger gels require 0.6 g agarose/100 ml 1xTBE. The gel should set for about 20-30 min. before use. 2. To set up the gels, put on gloves (ethidium bromide is a carcinogen!), and remove the tape from the ends of the gel. Place in the gel apparatus and cover the gel with 1xTBE electrophoresis buffer. Make sure the tray is oriented so that the wells are located closer to the black (negative) pole and away from the red (positive) pole. 3. Loading DNA samples: a. Each group of 2 or 3 should load a set of four samples. If you have two groups sharing a gel, you should run duplicate sets of samples. b. The samples have been prepared for you and contain the DNA sample mixed with loading buffer. Load 10 μl of each sample/well. Lane 1 2 3 4 Sample Suspect 1 Suspect 2 Suspect 3 Evidence 4. After the group’s samples are loaded, connect the cover and the electrodes so that the DNA samples will migrate toward the positive (red) pole. Insert the 1 red and black electrodes into the power supply with the power turned off. Turn the power on and adjust the voltage to 100 volts. Carry out the electrophoresis for about 1 hour or until the blue dye front has migrated one half to two thirds the distance down the gel. 5. Your gel will be photographed using the imaging workstation. Be sure to provide the instructor with information about your gel and which students worked together. You will be provided with a color printout; images can also be emailed at your request. Image files are rather large, so you might find it easiest to open a word file, insert the gel file as a picture, then resize the picture to approximately its normal size. Worksheet DNA Testing Lab (20 points) Worksheet due Wed. May 9, 2007 in class. Analysis of results: 1. Provide a picture of the gel electrophoresis result you obtained in lab. Label the lanes and attach to this assignment. 2. Based on the results, which suspect’s DNA pattern matchs that of the evidence at the crime scene? 3. a. Prepare a table of standard and unknown fragments. The fragment sizes from lane 1 (Suspect 1) are given. Measure the distance each standard fragment (in cm or mm) migrated using a centimeter ruler. Use this to construct a standard curve on semi-log graph paper. Be sure to label the axes of the graph. Lane 1:Suspect 1 Lane 2:Suspect 2 Lane 3:Suspect 3 Lane 4:Evidence distance base distance base distance base distance base pairs pairs pairs pairs 23,000 9400 6000 4400 (faint) 2200 2000 560 125 (usually not seen) 2 b. Describe some specific differences in the DNA banding pattern of the suspects whose DNA does not match the DNA pattern of the evidence. For full credit, use particular band sizes in your explanation. 4. Compare the DNA patterns of suspect 1 and suspect 2. a. Determine the sizes of any fragments that significantly differ between these two individuals, using the size information from question 3. b. How related are suspect 1 and suspect 2 likely to be, based on their DNA patterns? 3 5. a. Select one of the following methods or applications or another technology relevant to the study of human genetics or population genetics and briefly research it, using the web site of the European Initiative for Biotechnology Education, your textbook, and/or any other resources you choose. Topics: RFLP analysis PCR-based DNA profiling PCR-based DNA tests RAPD analysis SNP analysis Microarrays (DNA chips) Positional cloning Pyrosequencing Real-time PCR Reverse Transcriptase PCR RNA interference Transgenic mice Fluorescent in situ hybridization (FISH) Allele specific oligonucleotide hybridization Northern blot Spectral karyotype analysis (SKY) Taq-Man assay b. In an answer of one to two pages at most, in your own words define or briefly explain what the method or application entails, describe how it might be carried out, and provide a specific example of what you might learn from it or how it might be applied (historical examples are fine). Your answer should be neatly handwritten or computer printed, double-spaced, and enclosed with your worksheet. Be sure to list the references you consulted. For web sites, give the URL. For text or journal sources, give author, title, year, volume (journal), and pages. 4