A partnership between the UAB Center for Community Outreach Development and McWane Center DNA: A Person s Ultimate Fingerprint This project is supported by a Science Education Partnership Award (SEPA) from the National Center for Research Resources, National Institutes of Health. Revised 10-Sep-04
DNA: A Person s Ultimate Fingerprint The experiment described here is designed to allow students to examine individual variations in the human genome. Using polymerase chain reaction (PCR), a fingerprint of each member of the class will be made. DNA fragments generated by PCR will be separated by gel electrophoresis and analyzed for polymorphisms. Students will examine a single locus on chromosome 1, which is composed of non-coding repeats that vary in number from 14 to 41 in the human population. The experiment will allow students to isolate their own DNA from cheek cells, carry out PCR reactions and analyze the results of the PCR by gel electrophoresis. 2
Student Guide Human DNA Fingerprinting by Polymerase Chain Reaction In this experiment, polymerase chain reaction (PCR) is used to amplify a short nucleotide sequence from chromosome 1 to create a personal DNA fingerprint. Although the DNA from different individuals is more alike than different, there are many regions of the human chromosomes that exhibit a great deal of diversity. Such variable sequences are termed polymorphic (meaning many forms) and provide the basis for genetic disease diagnosis and forensic/paternity testing. Many DNA polymorphisms are found within the estimated 90% of the human genome that does not code for protein. A special type of polymorphism called a VNTR (variable number of tandem repeats) is composed of a certain DNA sequence that is repeated and repeated and repeated, with each repeat lying adjacent to the next one. Chromosome 1 contains a VNTR called D1S80, which has a repeat unit of 16 base pairs. At the D1S80 locus, most individuals have alleles containing between 14 and 41 repeats, which are inherited in a Mendelian fashion on the maternal and paternal copies of chromosome 1. 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 resuspended with the resin Chelex, which binds metal ions that inhibit the PCR reaction. The cells are lysed by boiling and centrifuged to remove cell debris. A sample of the supernatant containing chromosomal DNA is combined with a buffered solution of heat-stable Taq polymerase, two oligonucleotide primers, the four deoxynucleotide building blocks of DNA, and the cofactor MgCl 2. The PCR mixture is placed in a DNA thermal cycler and taken through 30 cycles consisting of: 1 minute at 94 C chromosomal DNA is denatured into single strands 1 minute at 65 C primers anneal to their complementary sequences on either side of the D1S80 locus via hydrogen bonds 1 minute at 72 C Taq polymerase extends a complementary DNA strand from each primer The primers used in the experiment bracket the D1S80 locus and selectively amplify that region of chromosome 1. Following PCR amplification, student alleles are separated according to size using agarose gel electrophoresis. After staining with ethidium bromide, one or two bands are visible in each student lane, indicating whether an individual is homozygous or heterozygous for the D1S80 locus. Different alleles appear as distinct bands each composed of several billion copies of the amplified allele. A band s position in the gel indicates the size (and number of repeats) of the D1S80 allele: smaller alleles move a longer distance from their origin, while larger alleles move a shorter distance. 3
Laboratory Procedure Procedure A. Isolation of cheek cell DNA 1. Use a permanent marker to label your name on a test tube containing saline solution and on two clean 1.5 ml microfuge tubes. 2. Pour all of the saline solution (10 ml) into your mouth and vigorously swish for 10 20 seconds. 3. Expel saline solution back into tube. 4. Using a P1000 micropipet, remove 1.5 ml of saline solution, now containing cheek cells, and add to one labeled microfuge tube. 5. Place a sample tube, together with other student samples, in balanced configuration in a microfuge and spin for 5 minutes at half maximum speed. 6. Carefully pour off supernatant into original test tube. Take care not to disturb cell pellet at the bottom of the microfuge tube. Remove any excess sup with a P100 or a tightly wrapped Kimwipe. Ask your facilitator for help! 7. Set micropipet to 500 µl. Draw Chelex suspension in and out of pipet tip several times to suspend resin beads. Then, before resin settles, rapidly transfer 500 µl of Chelex to the tube containing your cell pellet. 8. Resuspend cells by pipetting up and down several times. Examine against light to confirm that no visible clumps of cells remain. 9. Place your sample in a 95 C hot block for 10 minutes. Remove your tube from hot block and allow it to cool for a minute. 10. Place your sample tube in a balanced configuration in microcentrifuge, and spin for 30 seconds at maximum speed. 11. Use a fresh tip on the P100 micropipet to transfer 50 µl of the clear supernatant to the second labeled microfuge tube. Take care not to pick up Chelex/cell debris from the bottom of the tube. Discard tube with pellet of cells and Chelex. Begin Procedure B now (or store samples on ice until ready for Procedure B). 4
Procedure B. Set Up PCR Reaction: 1. Use permanent marker to label the cap of a PCR tube with your initials. Add the following reagents to the 0.2 ml PCR reaction tube: 37 µl Master Mix, (containing water, 10X Buffer and dntps) 3 µl D1S80 primers 5 µl cheek cell DNA 5 µl Taq polymerase (1u/µl) (Your facilitator will add the Taq. Make sure you watch it go in you PCR tube.) You should have a total reaction volume of 50µl. 2. Carefully close cap to PCR tube. Mix reagents by gently tapping tube bottom on lab bench. 3. Store your sample on ice or in the refrigerator until ready for amplification along with other student samples. 4. Program and start thermal cycler with a step file: (Your facilitator will help you do this!) Reaction volume is 50 µl. Hold 94 C 5 minutes 30 94 C 1 minute cycles 65 C 1 minute 72 C 1 minute Holds 72 C 10 minutes for final file 4 C Hold at end of run Prepare an agarose gel while your PCR reaction is in progress. Procedure C: Preparation of 1.5% Agarose Gel 1. Your facilitator will show each group how to prepare their 1.5% agarose gels, in 1x TAE buffer. Weigh 0.600 g agarose and transfer to an erlenmeyer flask. Add 40 ml 1X TAE buffer. Melt agarose in microwave or on a hot plate, swirling frequently. Let the agarose cool to the touch (but not solidify). Then add 5 µl of Ethidium Bromide stock (2 mg/ml) to agarose. Pour the agarose into a prepared gel casting tray. 5
NOTE FACILITATORS SHOULD RESTRICT HANDLING OF ETHIDIUM BROMIDE SINCE IT IS A HEALTH HAZARD. Procedure D. Agarose Gel Electrophoresis 1. Use permanent marker to label a clean 1.5 ml tube 2. Transfer 20 µl of your PCR sample to the labeled tube 3. Add 4 µl of loading dye to the PCR sample. Close tube and mix by tapping tube on bottom of lab bench or by pulsing in a microcentrifuge. 4. Add 20 µl of the PCR/loading dye sample into your assigned well of the 1.5% agarose gel. Expel any air in the tip before loading and be careful not to punch the tip of the pipet through the bottom of the sample well. 5. In each gel, run one lane with a DNA marker. Load 10 µl DNA marker into the gel as described above. The DNA marker contains a ladder of DNA fragments ranging in size from 100 bp to 2100 bp. Sizes are 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, etc. The 500 bp, 1000bp, and 2100 bp bands are especially bright and easy to locate. The size of your PCR product can be estimated by comparing its position to the DNA marker. 6. Electrophorese agarose gels at 150 volts for 35 minutes to one hour. Adequate separation will have occurred when the bromophenol dye front has moved at least 50 mm from wells. 7. Following electrophoresis turn off power supply, disconnect power leads, remove gel, and place on the UV transilluminator to observe and take photos of gel. Wear gloves at this step. RESULTS AND DISCUSSION 1. Examine the photograph of the stained gel containing your sample and those from other individuals. Orient the photograph with the sample wells at the top. First, look for a diffuse (fuzzy) band of primer dimer that might appear toward the bottom of the gel, at the same position in each lane. Primer dimer is not amplified human DNA, but is an artifact of the PCR reaction that results from primers amplifying themselves. Excluding primer dimer, interpret the allele bands in each lane of the gel: a. No bands visible. This usually results from an error during sample preparation, such as losing the cheek cell pellet or failing to resuspend Chelex beads prior to transferring solutions between test tubes. b. One band visible. The simplest explanation is that the individual is homozygous at the D1S80 locus, having inherited the same allele on maternal and paternal chromosome 1. However, since the samples were electrophoresed on agarose 6
gels, then it is more likely that the individual is, in fact, heterozygous, but the two alleles are so similar in size that they cannot be resolved (separated) in this gel system. Another possibility is that a larger allele (with many repeats) has failed to amplify efficiently. c. Two bands visible. The individual is heterozygous at the D1S80 locus. Often, the larger allele amplifies less efficiently and appears less intense than the smaller one. d. Three or more bands visible. The two brightest bands are likely the true alleles. Additional bands may occur when the primers bind nonspecifically to chromosome loci other than D1S80 and give rise to additional amplification products. 2. Population studies have identified 29 alleles at the D1S80 allele, and estimate that 90% of individuals are heterozygous at this locus. Determine the number of different alleles represented among your classmates and the percent of heterozygous individuals. How does your class data compare with that of the general population? What reasons can you give for differences? 3. Based on your results, do you think this protocol could be used to link a suspect with a crime or establish a paternity relationship? Why do you think so? How could you modify the experiment to improve its ability to positively identify individuals? 7
WORKING WITH YOUR DATA A. Analysis of D1S80 PCR products using agarose gel electorphoresis The figure below is an example of an agarose gel containing DNA from four students PCR reactions. In one lane there is a DNA marker; the other lanes contain PCR products for the D1S80 locus. Use the DNA marker to approximate the size, in base pairs, of the PCR products in each sample lane. For example, in the diagram below, the PCR products in lane 1 can be approximated at 640 base pairs (bp) and 320 bp. In lane 2, we see a single band at 400 bp. We can conclude from the data that the individuals in lane 1 and lane 2 are heterozygous and homozygous, respectively, with respect to the D1S80 locus. How would you interpret lanes 3 and 4? M 1 2 3 4 5 6 7 8 1000 700 500 400 300 200 100 8
After you have determined the size of the DNA bands on the gel, you can calculate the number of repeats at the D1S80 locus. But, first, look at an example for calculating the size of the PCR product for a given allelle. An allele composed of 17 repeats is used in this example. Notice that repeat unit 1 has 14 bp and the remaining repeat units have 16bp. However, this 2 bp difference doesn t change the number of repeats found at D1S80 during analysis, therefore we will assume repeat 1 has 16 bp in the following formulas. 5 1 TCAGCCCA-AGG-AAG 2 ACAGACCACAGGCAAG 3 GAGGACCACCGGAAAG 4 5 6 GAAGACCACCGGAAAG GAAGACCACCGGAAAG GAAGACCACAGGCAAG 7 8 GAGGACCACCGGAAAG GAGGACCACCGGCAAG 10 11 GAGGACCACCAGGAAG GAGGACCACCAGGAAG 9 GAGGACCACCGGCAAG 12 GAGGACCACCGGCAAG 13 14 15 GAGGACCACCAGGAAG GAGAACCACCAGGAAG GAGGACCACCAGGAAG 16 GAGGACCACCAGGAAG 17 GAGGACCACTGGCAAG 3 The length of D1S80 alleles can be calculated as follows: 115 bp upstream (region from "left" primer to repeat unit 1) + (total number of repeats ) x 16 base pairs + 32 bp downstream (region from last repeat to right primer) length of D1S80 in bp 5 upstream D1S80 downstream 1 17 3 9
In the example above, the size of the PCR products expected would be: 115 + (17 x 16) + 32 = 419 B. Calculate the number of repeats at your D1S80 loci To determine the number of repeats from the size of the bands on a gel, work backwards. Number of repeats = (# of BP estimated from Gel (115+32)) 16 Or to make it really simple Number of repeats = (# of BP estimated from Gel 147) 16 In the gel on page 8, lane 1 contains a PCR product of 640 bp and 320 bp. Thus, the number of repeats would be: ((640-147) / 16) = 30.81, or ~ 31 repeats. The number of repeats for 320 bp would be: ((320-147) / 16) = 10.81, or ~11 repeats. Calculate the number of repeats for the 400bp band: ((400-147) / 16) = repeats Now, calculate the number of repeats at your D1S80 loci. Fill in the box with the estimated size of your D1S80 locus and solve the equation below: (if you are heterozygous, you will have to do this twice!) Number of repeats = -147 ( ) 16 Finally, determine the size of the PCR products and the number of repeats for your DNA and the DNA of the other persons working at your table. Record the information in the table on the next page. 10
lane name size of DNA (base pairs) #of repeats homozygous or heterozygous Label and tape the picture of your gel in the space below. 11