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Lab 10 - PCR-Based Alu-Human DNA Typing Introduction: This lab is a modification of an exercise developed by Edvotek (Edvotek, 2003). It is a very interesting experiment designed to isolate human DNA and compare DNA polymorphisms between individuals. The human genome consists of 2.9 billion base pairs of DNA. Of this total, only about 5% consists of exons which code for protein. Introns and other noncoding sequences make up the remainder; although some of these sequences may possess undiscovered functions, most appear to have none. Many of these noncoding sequences appear to be self replicating as they have been found to be repeated hundreds or thousands of times throughout the genome. These repetitive sequences have been termed "selfish" or "parasitic" DNA, as they often appear to possess no function except for their own reproduction. These repetitive elements account for more than 20 percent of the human genome. In 1979, it was discovered that human DNA contains one of these repetitive elements, 300 base pairs in length, repeated hundreds of thousands of times. Copies of this element often contain a recognition site for the restriction enzyme AluI, and were subsequently named Alu elements. Although Alu elements have been found in exons, most exist in introns and other non-coding regions. When Alu sequences do insert into protein-coding regions, disruption of the gene usually results, often causing harm to the organism. Alu sequences replicate through an RNA intermediate which is then copied into a double-stranded DNA segment called a retrotransposon. The details of this process are not well understood. The retrotransposon then inserts, probably randomly, elsewhere in the genome. It is also theorized that most Alu sequences are incapable of replication and that only a small number of "master genes" are duplicated to form new elements. Although all humans (and other primates) possess hundreds of thousands of Alu elements, variations in their placement may occur in specific DNA regions. DNA sequences which vary between individuals are known as polymorphisms. For example, one person may possess an Alu insertion at a specific DNA locus, while another individual lacks that insertion. Furthermore, the insertion may be present or absent on each homologous chromosome; these Alu insertions are called dimorphic. One such dimorphic Alu sequence is found on a section of chromosome 16 known as the PV92 locus. This section of DNA is 700 nucleotides in length. Insertion of the 300 base pair Alu element results in a length increase to 1000 base pairs. One may test whether a person possesses an Alu insertion at the PV92 locus by amplification of the locus using the polymerase chain reaction (PCR). If a person is homozygous for the insertion, a gel of the PCR product will result in a single band at 1000 base pairs (Figure 1A). If a person is heterozygous, i.e., possesses the insertion on one chromosome 16 homologue but not the other, two bands will be present following PCR. One band will be 700 base pairs and the other will be 1000 (Figure 1B). If a person lacks the insertion on either chromosome homologue, that person is said to possess the null genotype and PCR will result in only one band at 700 base pairs (Figure 1C). To purify DNA for this type of analysis, almost any tissue or body fluid (except urine) may be used. The most common sources of human DNA are blood, hair, and saliva. The cells must be treated (lysed) to release their DNA into solution. Following lysis, the cells are often resuspended in a chelating agent, which removes cellular cations that inhibit PCR. PCR was invented in 1984 by Kary Mullis who was awarded a Nobel Prize for his work in 1994. The enormous utility of PCR is based on its ease of use and its ability to amplify DNA. The PCR process (Figure 2) uses an enzyme known as Taq polymerase. This enzyme is purified from a bacterium originally isolated from hot springs and is stable at very high temperatures. Also included in the PCR reaction mixture are two (15-30 nucleotide) synthetic oligonucleotides , known as "primers" and the extracted DNA template also known as the target DNA.
In the first step of the PCR reaction, the target complimentary DNA strands are melted (separated from each other) at 94C, while the Taq polymerase remains stable. In the second step, known as annealing, the sample is cooled to 65C to allow hybridization of the two primers to the two strands of the target DNA. In this experiment, the target is the PV92 locus in the extracted DNA. In the third step, the temperature is raised to 72C and the Taq polymerase adds nucleotides to the primers to complete the synthesis of the new complementary strands. These three steps - denaturation, annealing, and DNA synthesis - constitute one PCR "cycle".This process is typicality repeated for 30-40 cycles, amplifying the target sequence exponentially (Figure 2). PCR is performed in a thermal cycler, which is programmed to rapidly heat, cool and maintain samples at designated temperatures for varying amounts of time. In this experiment, each student will extract his/her DNA from cheek cells and amplify DNA at the PV92 locus by PCR. The PCR product(s) will then be examined on agarose gels to determine whether the student is homozygous (+/+), heterozygous (+/-), or null (-/-) for an Alu insertion at the locus. Objectives of this experiment are the isolation of human DNA and the comparison of DNA polymorphisms between individuals by PCR amplification and gel electrophoresis.
Procedure: I. Isolation of Cheek Cell DNA The first part of the experiment involves the isolation of DNA from cheek cells. This experiment can alternatively be performed using DNA isolated from hair. As with most molecular procedures, gloves and goggles should be worn routinely throughout the experiment as good laboratory practice. 1. Obtain cells by swabbing the inside of the mouth with a cotton-tipped applicator. Twirl the applicator while vigorously swabbing both cheeks, between the gum line and under the tongue . 2. Place the cotton head in 2 ml of PBS (in a labeled 15 ml conical tube) and twirl back and forth vigorously for 30 seconds to dislodge cells. Press the cotton head against the walls of the conical tube to squeeze out as much liquid as possible. 3. Using a fresh applicator, repeat steps 1 and 2. Twirl the applicator to add cells to the same tube containing the 2 ml of PBS. 4. With a calibrated transfer pipette, transfer 2 ml of the cells in PBS to a 2 ml screw cap microtest tube, be sure to label your tubes. 5. Spin at 5000 - 6000 g in a microcentrifuge (with appropriate counterbalance) for 1 minute. Important: Check to see that the buffer is clear (not cloudy) and that there is a visible white pellet (approximately 7-8 mm in length). If the buffer is cloudy with little or no pellet, spin the tube(s) for an additional 30 - 60 seconds. If the buffer is clear with little or no pellet, obtain another swab and repeat steps 1-5. 6. Pipette off the supernatant, using care to avoid discarding the pellet. 7. Mix the tube of chelating agent by inverting it several times. The chelating agent is used to bind cations released by the cells that inhibit PCR. a. Continue to mix the chelating agent with a calibrated transfer pipette by pipeting up and down several times. b. Next, quickly add 100 l of the chelating agent to the tube containing the pellet. 8. Resuspend the pellet in chelating agent by pipetting up and down several times, or by vortexing gently. Check to see that the pellet is fully suspended. 9. Lyse the cells by placing the tube in a boiling waterbath for 10 minutes. 10. Allow the tube to cool for 2 minutes. Vortex for 10 seconds or tap the tube vigorously to mix. 11. Spin the tube in a microcentrifuge for 2 minutes to pellet the cell debris, leaving DNA in the supernatant. 12. Transfer 20-50 ul of the supernatant to a clean 1.5 ml tube, using care to avoid disturbing the pellet. It is very important not to take over even one of the beads, if you do, your PCR amplification will not work, as the bead will take up all the Mg need for the reaction. 13. Place the sample (supernatant) on ice.
THIS IS AN OPTIONAL STOPPING POINT The supernatant may be stored at -20C until the experiment is continued.
II. Amplification of the PV92 Locus Now that we have our DNA samples isolated, we can amplify our PV92 locus. It is important to remember in this part of the experiment that sample volumes are very small. For liquid samples, it is important to quick spin the tube contents in a microcentrifuge to obtain sufficient volume for pipeting. Spin samples for 10-20 seconds at maximum speed. 1. Label the tube containing the PCR reaction bead (PCR bead contains Taq DNA polymerase, the four deoxytriphosphates, Mg+2 and buffer) with your initials. Tap the reaction tube to assure the reaction pellet is at the bottom of the tube. 2. To prepare the PCR reaction mix tube, add the following to the bead: PV92 primer solution 20.0 ul Cheek cell DNA (supernatant) 5.0 ul 3. Gently mix the PCR reaction tube and quick spin it in a microcentrifuge to collect all the sample at the bottom of the tube. Next transfer your sample to the smaller microfuge tubes for the PCR machine. 4. Put the PCR tube through the cycles automatically in a thermal cycler. Process for 32 cycles. 94C for 30 seconds 58C for 30 seconds 72C for 30 seconds 5. After the 32 cycles are completed, add 5 ul of 10x Gel Loading Solution to the sample and store on ice.
OPTIONAL STOPPING POINT The samples can be held in the thermal cycler at 4C or frozen after addition of 5 ul of 10x Gel Loading Solution until ready for electrophoresis.
III: Separation of PCR Reactions by Electrophoresis In the third part of the experiment we will run the products of the PCR reaction on gels and analyze the results using the following procedures: 1. Close off the open ends of a clean and dry gel bed (casting tray) by using rubber dams. Place a rubber dam on each end of the bed. Make sure the rubber dam sits firmly in contact with the sides and bottom of the bed. 2. Place a well-former template (comb) in the first set of notches nearest the end of the gel bed. Make sure the comb sits firmly and evenly across the bed. A number of different sized gels can be used in this procedure, but better resolution of the PCR products is seen with larger-sized gels, like a 7 x 15 cm gel. 3. This experiment requires a 1.5% gel. If we are using the smaller sized gels, mix 0.6 gms of agarose with 40 ml of 1X electrophoresis buffer. Swirl to disperse clumps. 4. Heat the mixture to dissolve the agarose powder. The final solution should be clear (like water) without any undissolved particles remaining. a. Cover flask with plastic wrap to minimize evaporation. b. Heat the mixture on High for 1 minute. c. Swirl the mixture and heat on High in bursts of 25 seconds until all the agarose is completely dissolved. 5. Cool the agarose solution to 55C with gentle swirling to promote even dissipation of heat. If detectable evaporation has occurred, add distilled water to bring the solution up to the original volume as marked on the flask in step 5. 6. Pour the cooled agarose solution into the bed. Make sure the bed is on a level surface. 7. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 20 minutes. 8. Carefully and slowly remove the rubber dams. Be careful not to damage or tear the gel when removing rubber dams. A thin plastic knife, razor or spatula can be inserted between the gel and the dams to break possible surface tension. 9. Next, remove the combs by slowly pulling them straight up. Do this carefully and evenly to prevent tearing the sample wells. 10. Place the gel (on its bed) into the electrophoresis chamber, properly oriented, centered and level on the platform.Caution! 11. Fill the chamber of the electrophoresis apparatus with the required volume of diluted buffer. 12. The agarose gel is sometimes referred to as a "submarine gel". This is because the gel issubmerged under buffer for sample loading and electrophoretic separation. Make sure the gel is completely covered with buffer. 13. Heat the 200 bp DNA ladder (C) and PCR samples for two minutes at 50C in hotblock. Allow the samples to cool for a few minutes. 14. Load 27 ul of your 30 ul sample. Note, it is important to record the position of your sample in the gel. Also, during electrophoresis, the DNA samples migrate through the agarose gel towards the positive electrode. Before loading the samples, make sure the gel is properly oriented in the apparatus chamber. 15. Set the power source at 120 volts and run the electrophoresis for the length of time as determined by your instructor (1.5-2.0 hours optimum). When current is flowing properly, you should see bubbles forming on the electrodes. 16. Allow the tracking dye to migrate 4.5 cm. (small gel) or 8 cm. (large gel) from the wells for adequate separation of the DNA bands. Terminate the electrophoresis before the tracking dye moves off the end of the gel. 17. After electrophoresis is completed, place the gel on a piece of plastic wrap on a flat surface. Moisten the gel with a few drops of electrophoresis buffer. Wearing gloves, place the unprinted side of the DNA InstaStain/EtBr sheet on the gel. 18. Firmly run your fingers over the entire surface of the DNA InstaStain/EtBr. Do this several times. 19. Place the gel casting tray and a small empty beaker on top. This will ensure that the InstaStain sheet maintains good contact with the gel surface. Allow the DNA InstaStain/EtBr to sit for 15 minutes. 20. After 15 minutes*, remove the sheet of DNA InstaStain/EtBr. Transfer the gel to a short wave (300 nm) ultraviolet transilluminator for viewing the DNA. Be sure to wear UV protective goggles. *If bands appear faint, gels may take longer to stain. Repeat the staining procedure and increase staining time and additional 15 minutes. If all the bands are not visible, the staining procedure can be repeated. Assingment: 1. What does your gel look like and what do your results mean? 2. Compare your Alu genotype with those of your classmates. Did anyone else have a similar result? If so, what are some possible explanations?
Materials: Thermal Cycler (EDVOTEK catalog #532 is highly recommended) Horizontal Gel Electrophoresis Apparatus D.C. Power Supply Microcentrifuges UV Transilluminator UV Photodocumentation System (optional) Eppendorf Micropipeters and tips both 10-100 ul and 0.5-10 ul sizes Hot Block Hot Gloves Distilled or Deionized Water Pipette Pump 250 ml Flasks or beakers Ice buckets and ice UV Safety Goggles Disposable laboratory gloves UltraSpec-Agarose Electrophoresis Buffer 10x Gel Loading Buffer DNA InstaStain/EtBr sheets Microcentrifuge Tubes (2 ml) Microcentrifuge Tubes (1.5 ml) Screw cap conical tubes (15 ml) Cotton Swabs Calibrated transfer pipets
Contents Storage A. PV92 primer mix -20C Freezer B. Qualified water -20C Freezer C. 200 base pair ladder -20C Freezer D. Chelating Agent Room Temperature E. 10x PBS Room Temperature F. PCR Tubes with Beads which contain: Room Temperature
a. dNTP Mixture b. Taq DNA Polymerase Buffer c. Taq DNA Polymerase d. MgCl2 Note, sample volumes are very small. It is important to centrifuge all biological samples in a microcentrifuge for 10-20 seconds at maximum speed to recover materials required for this experiment.
References: EDVOTEK, Inc. 2003, EDVO-Kit # 333, 14676 Rothgeb Drive, Rockville, MD 20850 |