1 Biology 423L Nov. 6/7 Genetics in Forensic Science: Human DNA Fingerprinting Report due Nov. 21 Readings: Hartwell et al. pp. 297-302, 374-387. Nakamura Y., Carlson, K. Krapco, and R. White 1988. Isolation and mapping of a polymorphic DNA sequence (pmtc118) on chromosome 1p. Nucleic Acids Research 16: 9364. Objective: In this lab exercise, the polymerase chain reaction (PCR) will be used to amplify a locus from your own DNA to create a personal fingerprint. Although DNA from different individuals is 99.9% alike, there are variable regions that are polymorphic and provide the basis for genetic disease diagnosis, forensic identification, and paternity testing. You will work in groups as usual but you will each extract and amplify your DNA. Introduction: In this experiment, students will amplify a non-coding region of chromosome 1 containing the VNTR (microsatellite) sequence designated pmct118. It has a repeat unit of 16 base pairs. Most individuals have between 14 and 40 copies of the repeat on each of their copies of chromosome 1. An individual s two copies of chromosome 1 usually have different numbers of copies, as do the chromosomes from two different individuals. The different versions of the pmct118 polymorphism are referred to as alleles and are inherited in a Mendelian fashion. The pmtc118 locus has no known relationship to any disease state or any other phenotype. The source of template DNA is a sample of several thousand cheek cells obtained by saline mouthwash (bloodless and noninvasive). The cells are collected by centrifugation and heated in a buffered solution containing beads of the resin Chelex. Chelex binds (chelates) metal ions that act as PCR inhibitors and as catalysts in the breakdown of DNA at high temperatures. Lysis of the cells and release of DNA is accomplished by a boiling water bath and the alkalinity of the Chelex suspension. This is a "quick and dirty" 1
2 extraction method (the DNA will probably be unamplifiable after a year at -20 o C). PCR is then performed with the DNA-containing supernatant. You can visit the DNA learning center at http://dnalc.org. Click on Resources then [Biology] Animation Library to view of download animations on PCR and DNA fingerprinting using VNTRs Materials: Gloves Tip disposal Boiling water bath (Hot plate and large glass beaker) Floating tube racks Forceps Microfuge Capped 15 ml tubes 1.5 ml l tubes Paper cups Eppendorf pipet aids P1000, P200, P20 Blue and yellow tips (autoclaved) Kimwipes Ice buckets TA needs an ice bucket to store samples when ready for PCR. O.9 % Saline solution (autoclaved) in 15 ml capped centrifuge tubes at 10 ml per tube. Water (autoclaved) for Chelex solution. Chelex resin 10% slurry in autoclaved H 2 O PCR reaction beads in 500 µl tubes (1 for each person) PCR cycler 2% Agarose gel (1/2 nusieve: ½ normal agarose with ethidium bromide) Gel electrophoresis apparatus DNA size markers (pbr322 digested with BstN1, sizes are: 1857 bp, 1058 bp, 929 bp, 383 bp and 121 bp) use 20 µl per gel.. Video camera Method: Assuming the protocol is rigorously followed, amplification problems in this lab probably relate to poor oral hygiene and inhibitors present in food particles and bacteria. Before you come to class, make sure there are no food particles in your mouth. (If possible, brush your teeth and rinse thoroughly with water.) Wear Gloves to avoid contamination of the solutions with cells from your hands 2
3 1. Label a 15 ml tube with a number assigned by the TA. The TA will give each person in the class (both sections) their own identification number (on the PCR bead tube see step 4). Each person will prepare one sample of their own DNA. 2.Pour 10 ml of saline solution (0.9% NaCl) into a cup, then into your mouth. Swish it vigorously around your mouth for 10 seconds. Then expel the fluid back into the cup. 3. Pour the solution back into the 15 ml tube. 4. Place your tube, in a balanced configuration with others, in a clinical centrifuge and spin for 10 minutes at full speed. At this time, get two 1.5 ml sterile eppendorf tubes per person and a 0.5 ml PCR bead eppendorf tube per person. Label both with your identification number. A 1.5 ml tube will contain your buccal DNA sample. At the end of the lab period your TA will keep the unused part of this sample in case they need to repeat the PCR reactions. The 0.5 ml tube will contain your PCR reaction. You will give this to your TA at the end of the lab so they can run the reactions in the PCR cycler. 5. Back to the DNA prep with the 15 ml tube after centrifugation. Being very careful not to disturb the pellet, carefully remove the supernatant by pouring it out of the tube into your paper cup. Important: Before you turn the tube back up, remove the last bit of liquid from the side with a Kimwipe. You can now dispose of the solution in the paper cup by pouring it down the sink. 6. Use your P1000 pipetman to transfer 500 µl of Chelex slurry into your test tube. Be sure the Chelex resin is well re-suspended in the water before pipetting. 7. Draw the liquid in the tube up and down with your pipetteman and the blue tip several times to re-suspend the cell pellet. Be sure the pellet is completely re-suspended before proceeding. 8. Transfer 500 µl of the Chelex-cell suspension to one of the 1.5 ml tubes you have labeled earlier. Be sure the Chelex beads remain well suspended in the transferred sample. Use your pipetteman to pipette up and down to be sure the cells and Chelex are well suspended. 9. Place your sample in a floating tube rack in a boiling water bath for 10 minutes. The TA will collect the tubes and put them in the boiling water. After your tube has been incubated 10 minutes, use forceps to remove it and cool the tube on ice for 30 seconds. 10. Place your tube in a balanced configuration with others in a microcentrifuge, and spin at full speed for 30 seconds. 3
4 11. Set your P200 pipetteman for 200 μl and transfer 200 μl of the clear supernatant to a clean, labeled 1.5 ml eppendorf tube. Be very careful not to take any of the pellet. Store your sample on ice. 12. Dispose of the yellow tip in the container provided. PCR reactions Set up the PCR reactions as follows: 1. Use a pipettman with a fresh tip to transfer 22.5 µl of pmtc118 primer/loading buffer mix (red) to your labeled PCR tube containing a PCR bead. Tap the tube with a finger to dissolve the bead. 2. Use a fresh tip to add 2.5 µl of your DNA prep to the reaction tube, and tap to mix. Pool reagents by tapping on the lab bench. 3. Put your sample in an ice bucket for your TA to take to the thermocycler. Your PCR reaction will have the following final components: 10 mm Tris-HC1 (ph 9.0 at 25 o C) - provides optimal ph for enzyme 50 mm KC1 - maintains ionic strength for Taq 1.5 mm MgCl 2 - cofactor required by Taq 5 µm dntp's (1.25 μm of each) - datp, dgtp, dctp, dttp 5 µm of oligonucleotide primer 1.3 units Taq DNA polymerase - thermostable DNA synthesizing enzyme from Thermus aquaticus The PCR reactions will be run in a thermal cycler as follows: Your TA will run the thermal cycler. 1. 94 o C 4 min 2. 94 C 30 sec- denaturation of template 3. 65 C 30 sec- annealing of primers 4. 72 C 30 sec- synthesis of complimentary DNA strands (repeat from step2 x3o cycles) 5. 72 C for 10 minute final extension. 6. 10 C until samples are removed. For lab report: Your TA will use gel electrophoresis to analyze the PCR reactions on a 2% agarose gel in TAE buffer along with 20 µl of the pbr322-bstn1 size marker in one lane. The gels will contain the intercalator ethidium bromide which causes fluorescence of ethidium-dna complexes under UV light and hence allows the DNA bands to be visualized. The TA will electrophorese the samples at 100 volts until the creosol red dye in the mixture has moved to within one inch of the bottom of the gel. 4
5 They will take a photo of each gel with a ruler. Gels of the entire class s results will be posted on the web page. Questions and analysis of your amplified DNA Gels of the entire class s results will be posted on the web page. For your report, you will determine your own genotype for the pmtc118 alleles and calculate the frequency of each allele for the entire class (for both lab sections). Only count lanes with clear PCR results. Determine the proportion of people who are homozygous for each allele and heterozygous. Include the proportion of each of these genotypes in the results of your report. In order to distinguish the alleles, measure their size on the gel. Each band of a distinct size is a distinct allele. To generate a standard curve to measure the sizes of each band, plot the positions of the pbr322-bstn1 DNA ladder bands on a piece of semi-log paper. On the X axis (the linear axis), put the distance traveled from the well in centimeters (ruler will be on the photograph). On the Y axis (the logarithmic axis), put the size of the ladder band in base-pairs. The sizes of the bands are: 1857 bp, 1058 bp, 929 bp, 383 bp and 121 bp. When you plot all of these bands you should obtain something close to a line, because the relative mobility of a DNA molecule in an agarose gel is directly proportional to the log of its molecular weight. Include the standard curve in the report. Determine the approximate size of the PCR products in the gel by measuring the distance they migrated from the well and finding where this distance falls on your standard curve. Only determine the size of the two brightest and fastest migrating bands in each lane. Extra and fainter bands are likely to be artifacts. Using lanes with clear two band PCR product patterns, identify the sizes of the pmtc118 alleles in those lanes. How many different sized alleles are present in the population of your class? Make a table with the different size classes. What is the number of alleles of each size class? Add that to your table. What is the frequency of people in your class being homozygous for any allele? Add this to the table. Is there an allele that is more common than others? If so what is its size and frequency in the population of your lab group? Answer the following question at the end of your report with the title Question. The sizes of the PCR products of the pmtc118 locus in one family were 531 and 643 bp in the mother and 435 and 531 bp in the father. What are all possible fingerprints their children could have? 5