PCR AMPLIFICATION OF CHEEK CELL DNA

PCR AMPLIFICATION OF CHEEK CELL DNA LAB MBDNA3 From Chromosome 16: PV92 PCR Informatics Kit, Biotechnology Explorer, BioRad, 2004, and Isolation and P...
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PCR AMPLIFICATION OF CHEEK CELL DNA LAB MBDNA3 From Chromosome 16: PV92 PCR Informatics Kit, Biotechnology Explorer, BioRad, 2004, and Isolation and PCR Amplification of Human Chromosomal DNA, Lab 4a-4b, Science and Ethics of the Human Genome Research, Spring 2005, Prof. Joshua Corrette-Bennett, Westminster College.

INTRODUCTION DNA DNA is found in all living organisms. It contains the genetic code for making proteins – especially enzymes – that are necessary for the production of all other molecules in the cell. Thus DNA is the “master molecule” of the cell. It is responsible for passing traits from one generation to the next and allows species to adapt over time. While DNA is fairly consistent in structure and function in all cells and organisms, variations in the genetic code can and do exist. DNA exhibits variations known as polymorphisms. The majority of these polymorphisms occur within the base-pair sequences and range from single base-pair alterations (different base-pairs, deletions, insertions, etc.) to large insertions or deletions of DNA sequence (thousands of base-pairs). These variations can be analyzed using current biotechnology to reveal important information about an individual or groups of individuals. DNA Polymorphisms and the PV92 Locus Every human genome contains short repetitive interspersed elements (also known as SINEs). Each SINE consists of a relatively short sequence, anywhere from ten to a few hundred base-pairs in length, and exists at numerous places throughout the genome. These repeated sequences have been randomly inserting themselves throughout our genomes for millions of years. Over 44% of the human genome consists of repetitive DNA sequences! One such SINE is the Alu sequence, a 300 base-pair stretch of DNA sequence that exists at more than 500,000 places throughout the human genome! The origin and function of this SINE is not completely understood, but it does not code for any type of protein or RNA. We will analyze the Alu SINE at one specific location, called the PV92 locus, on Chromosome 16. Not everyone has the Alu repeat at this position of chromosome 16. This particular genetic locus is dimorphic (two forms). That is, the Alu element is either present or absent at this particular position on each chromosome 16. Some people may have the Alu repeat inserted at the PV92 locus on both chromosomes (homozygous +/+), some may not have the Alu repeat in either chromosome (homozygous -/-), and some may have the Alu repeat in one, but not the other (heterozygous). The presence or absence of the insert can be detected using the PCR reaction followed by agarose gel electrophoresis.

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PCR Amplification of Cheek Cell DNA

Figure 1. The presence or absence of the Alu insert within the PV92 locus on Chromosome 16.

PCR Amplification of DNA During the mid-1980’s, a creative scientist developed an elegant and powerful technique to generate millions of copies of a specific DNA sequence. This technique is called polymerase chain reaction amplification, or PCR amplification. It amplifies, or copies, a specified region of DNA sequence. This is particularly useful when there is very little sample to begin with, there has been some degradation of template, or when the amount and complexity of DNA sequence being searched is very high. PCR amplification produces many copies of target DNA in quantities sufficient for detection and analysis by agarose gel electrophoresis. PCR is most efficient at amplifying fragments of DNA that are a few thousand base pairs (bp) or less, but technology in this field is constantly improving and can now provide amplification of much longer pieces (up to 21,000 bp). PCR is analogous to photocopying: we start with an original (the desired DNA template or target sequence), use the appropriate machinery (a thermal cycler) and reagents (DNA polymerase, nucleotides, primers), and produce millions of identical DNA sequences, just as a Xerox machine can produce many copies of an original document. Reactions that occur in a PCR tube are equivalent to what occurs in the nucleus of a cell every time the cell replicates, or synthesizes, chromosomes in preparation for cell division. Many of the same components used by the cell are included in the PCR reaction, but the PCR tube contains only the bare minimum of components necessary for DNA synthesis. Primers are a critical component of DNA replication and thus PCR, providing specificity for copying the desired region. A primer is a short (12-50 base-pair), single-stranded sequence of DNA capable of pairing with DNA sequences on either side of the desired target sequence. Often primers are referred to as oligonucleotides. Two different primers are used for PCR. The primers should bracket the target region and direct the synthesis of new DNA strands in the direction of the other primer (towards each other). The primers provided for this PCR reaction are specific for DNA sequences that flank either side of the PV92 site on human Chromosome 16, regardless of whether the Alu insert is present or not (Fig. 2). If the Alu insert is present at the PV92 locus, the PCR reaction Westminster College – Science in Motion

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PCR Amplification of Cheek Cell DNA will amplify a piece of DNA that is 941 bp long. The absence of the Alu insert produces a 641 bp DNA fragment.

Alu

After PCR: Alu Alu Alu 941 bp

641 bp

Fig 2. The PV92 locus on Chromosome 16. The arrows represent the primers used in the PCR reaction. Two different size DNA fragments are produced with these primers, depending on whether the Alu insert is present (941 bp) or absent (641 bp).

Once the primers have bound to their complementary sequences, DNA polymerase uses each primer as a starting point for synthesis of a new DNA strand. In this way, the two strands of the double-stranded target region are bracketed by the two primers and replicated simultaneously. Template DNA must be included in the reaction. It can be obtained from many human tissues such as cheek cells, a hair follicle, skin cells, or blood. In theory, just a few cells or bits of tissues will supply enough DNA for the reaction but PCR requires more than just one or two strands of DNA template. While PCR is capable of amplifying crude DNA preparations and samples that have experienced some degree of degradation, DNA samples that have experienced significant levels of DNA degradation (e.g., samples that are millions of years old) do not make good templates. All four types of nucleotides (called nucleoside triphosphates; deoxyadenine [dA], deoxycytosine [dC], deoxyguanine [dG], and deoxythymine [dT]) are required for the reaction. These are the building blocks that will be used by DNA polymerase to construct the new DNA strands. Buffer is required to provide and maintain the correct environment for the DNA polymerase enzyme to function. Varying the concentration of the magnesium can also affect the efficiency of PCR, but we will be keeping the concentration constant. Taq DNA polymerase is the enzyme which performs the DNA synthesis. This is a special polymerase, isolated from a thermophilic bacterium (inhabits volcanic hot springs Westminster College – Science in Motion

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PCR Amplification of Cheek Cell DNA or deep sea thermal vents) bacteria. At high temperatures (70°C - 95°C), this type of DNA polymerase does not denature as rapidly as other enzymes so it does not lose its activity as rapidly. Try to imagine how well your body would function in a 70°C or 90°C water bath! As with any DNA polymerase, this enzyme joins individual nucleotides to the growing DNA strand, synthesizing DNA in one direction. The enzyme can survive multiple rounds of high temperatures used for denaturing double-stranded DNA. Not only can the enzyme survive higher temperatures, its peak activity occurs at higher temperatures (72°C). Temperature cycling is essential to PCR. After the template DNA and all reagents have been mixed, the cycling process of PCR begins. There are three different temperature steps to each cycle of PCR. One PCR Cycle Denature DNA Anneal Primers Synthesize New DNA

94ºC 60ºC 72ºC

The first step of the cycle requires very high temperature in order to completely denature the two strands of the template DNA. In the second step, the temperature is lowered to allow the primers to anneal to the template DNA. Primers that remain bound to their complementary DNA target sequence at higher temperatures are preferred because high annealing temperatures allow for greater specificity, referred to as stringency. The optimal annealing temperature for the primers provided with this kit is 60ºC. The primers annealed to the template form a short double-stranded region which directs the Taq polymerase to start synthesis at this site, thus targeting the amplified region. The final step of the PCR cycle is performed at 72ºC, the optimal temperature for DNA synthesis for the Taq polymerase. The PCR cycle shown above is programmed into the PCR Thermal Cycler and is repeated 40 times during the experiment. OBJECTIVE Each student will isolate DNA from cheek cells and use it as template DNA in a PCR reaction. The amplified DNA will be isolated on an agarose gel and analyzed. Each student will be able to determine if he/she is homozygous (+/+), homozygous (-/-) or heterozygous (+/-) for the Alu repeat.

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PCR Amplification of Cheek Cell DNA MATERIALS Day 1 1.5 mL snap-cap microcentrifuge tube (micro test tube) one screw cap micro test tube containing 200µL Instagene matrix 10 mL of 0.9% NaCl (saline) solution 200-1000 µL micropipettor & pipet tips 2-20 µL micropipettor & pipet tips sterile cotton swab sticks microcentrifuge waste beaker Kimwipes Forceps 2 water baths (56ºC & 100ºC) foam microcentrifuge tube holder refrigerator Day 2 0.2 mL microcentrifuge tubes containing 20 µL “Master Mix” ice and ice buckets 2-20 µL micropipettor & pipet tips PCR Thermal cycler Day 3 Electrophoresis chamber 1% TAE agarose gel 1x TAE electrophoresis buffer 2-20 µL micropipettor & pipet tips MMR (DNA size standards) (+/+) PV92 locus control (+/-) PV92 locus control (-/-) PV92 locus control individual PCR sample sample loading dye FastBlast agarose gel stain (1X) Gloves Staining trays Labeling tape and Sharpies

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PCR Amplification of Cheek Cell DNA PROCEDURES Day 1 DNA Isolation of Cheek Cells ►Under real research conditions, gloves would be worn at all times to prevent contamination of samples. We will not be adhering to this rule, as it makes the microcentrifuge tubes difficult to handle.◄ 1. Label with your initials: • one 1.5 mL snap-cap microcentrifuge tube (micro test tube) • one screw cap micro test tube containing 200µL Instagene matrix 2. There should be small cups containing 10 mL of 0.9% NaCl (saline) solution at each station. Select one of the cups and pour the yummy saline solution into your mouth, rinse vigorously for 45 seconds, then expel (yes, spit) the saline solution back into your cup. Food chunks will not make your experiment go well. If you have just eaten, rinse your mouth with water before starting. 3. Take the 200-1000 µL micropipettor, set to 1,000 µL (1 mL), place a fresh microtip onto the end, then transfer 1 mL of your saline solution/epithelial cheek cells into your labeled snap-cap micro test tube. Make sure each person uses a fresh microtip for the transfer process. Think about why this is important! 4. Take one of the sterile cotton swabs, dip the end you are not touching into the remaining expelled saline solution in your cup and use it to swab the inside of one cheek. Twirl the cotton swab vigorously while swabbing the cheek and gum area (don’t be timid!). Then dip the swab into the snap-cap tube with the 1 mL saline/cheek cell solution, twirling to dislodge any cells. Press the cotton swab up against the wall of the tube while twirling to squeeze out as many cells and as much liquid as possible. Repeat with a new cotton swab and the other cheek. 5. Place your micro test tube into one of the microcentrifuges. Be sure that your initials are clearly labeled. Spin the tubes for 2 min. Do not begin the spin until all the slots in the rotor have been filled! If all the slots are not filled, have your instructor check to be sure the rotor is balanced. 6. Once the centrifuge has completely stopped, remove your tubes. A match-head sized pellet of whitish cells should be visible. If you can’t see a pellet, carefully pour off (decant) the saline solution into a waste beaker without disturbing the pellet. Refill the micro test tube with another 1 mL of the saline/cheek cell solution and repeat the spin. 7. If a pellet is clearly visible, decant the saline solution into a waste beaker, being careful not to lose your pellet. Blot the tube briefly on a Kimwipe. It is OK to have a small amount of saline remain in the bottom of the tube, but it shouldn’t be more than 50 µL. Westminster College – Science in Motion

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PCR Amplification of Cheek Cell DNA 8. Snap the top of the tube shut and loosen the pellet by flicking the tube with your finger until clumps are no longer visible. 9. Using a 2-20 µL micropipettor set to 20 µL (plus a yellow microtip), transfer all of your resuspended cells to your labeled screwcap micro test tube containing the Instagene matrix. This may require repeated transfers. If the micropipettor is not sucking up your cells, then they are probably not resuspended and you should flick the tube a few more times. Make sure each person uses a fresh microtip for the transfer process. 10. Once you have transferred all of your cells, screw the cap tightly onto the tube and then flick the tube again to thoroughly mix the contents. 11. Place the tube in a foam micro test tube holder (make sure your initials are clearly labeled!) and incubate in a 56ºC water bath for 10 min. At the halfway time point (5 min), briefly remix the contents of the tube and quickly place the tube back in the water bath for the remaining 5 min. 12. After the 10 min at 56ºC, briefly remix the contents by tapping the side of the tube, put it back in the foam micro tube holder and place it immediately into the 100ºC water bath. Be careful not to touch the water with your hands! You will burn yourself! Incubate the tubes at 100ºC for 5 min. 13. Remove your tube from the 100ºC water bath and briefly mix the contents of the tube. Pellet the matrix/cell lysate by spinning the tube in the microcentrifuge for 10 min. As in Step 5, be sure the rotor is full or have your instructor be sure the rotor is balanced! 14. Remove your tube from the microcentrifuge and carefully store your sample in the refrigerator overnight. Day 2 PCR Amplification of the Target Region PV92 1. If your matrix/cell lysate sample has been refrigerated overnight, re-pellet the contents by centrifuging the tube for 5 min. 2. Your instructor will provide very small (0.2 mL) micro test tubes containing the correct amount (20 µL) of the “Master Mix”. The “Master Mix” contains the nucleotides, buffer and Taq DNA polymerase necessary for the PCR reaction. The thin walls of these tubes improve the efficiency of heat transfer to your sample during the PCR amplification procedure. But this also means the tubes are fairly delicate and pliable. Don’t squeeze them too hard! Each of you should take one of these tubes, label it clearly with your initials and keep it in the ice bath provided! 3. Using a 2-20 µL micropipettor, transfer 20 µL of the cell lysate from the Instagene screw cap tube into the PCR tube containing the Master Mix. ► When removing the

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PCR Amplification of Cheek Cell DNA lysate from the screw cap tube, be very careful NOT to transfer any of the Instagene matrix into the PCR tube. Remove the lysate from the TOP of the liquid layer in the Instagene tube. Your PCR reaction will not work if there is any matrix present.◄ Be sure to dispense the lysate into the very bottom of the 0.2 mL tube containing the Master Mix – this helps combine the two solutions. 4. Cap your tube and store it on ice until the instructor is ready to have you place your samples into the thermal cycler. Place your sample tube in a capless tube adapter and briefly centrifuge your sample (15 sec) so that everything is at the bottom of the tube. 5. When the instructor is ready, place all of the tubes from your group together in one row of the thermal cycler block. Be sure they are clearly labeled! (You might want to write down which row and what order the samples went in.) You are now ready to amplify! The reactions will run for 40 cycles or roughly 3 hours. Your instructor will provide you with the specifics of the PCR program. The PCR samples will be removed from the thermal cycler and stored at -20ºC until the class is ready to perform the agarose gel electrophoresis and analysis of the PCR products.

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PCR Amplification of Cheek Cell DNA Day 3 Agarose Gel Electrophoresis The instructor will hand out three PCR “positive control” samples to each group. One positive control represents amplified products from an individual who is homozygous for Alu inserts at the PV92 locus (+/+). The second control represents amplified products from an individual who is homozygous for no Alu inserts (-/-). And the last positive control represents amplified products from an individual who is heterozygous; one chromosome contains an Alu insert while the homologous chromosome does not (+/-). You will also be given one tube that contains loading dye (PV92 XC) and another that contains your DNA size standards (“MMR”). It is crucial that you keep track of these tubes and know what is required for each step. 1. Obtain your PCR amplified sample from the instructor. Place it in a capless tube adapter and spin in the microcentrifuge for a few seconds. If all the spaces in the centrifuge rotor are not filled, have your instructor check to be sure that the tubes are balanced. 2. Add 10 µL of PV92 XC loading dye to your PCR tube and mix well by flicking the tube. This dye serves a dual purpose, but it does not stain DNA. First, it increases the density of the sample so that the entire sample will sink into the agarose gel well and remain there. Second, it contains a dye that will migrate through the gel. The dye allows you to visualize the progress of electrophoretic separation. 3. You will be given a pre-cast 1% agarose gel still in the plastic casting tray. Carefully remove the tape from the ends of the casting tray and place the gel, still in the casting tray, into the electrophoresis chamber. Pour enough of the 1X TAE buffer into the electrophoresis chamber so that the gel is completely submerged. Remove the comb slowly by pulling straight up on it. You must do this carefully so that the bottoms of the wells do not rip. Orient the gel so that the wells are near the negative (black) terminal. 4. Your instructor will demonstrate how to load the agarose gel wells by loading the Molecular Mass Ruler (MMR) sample for you. There are several things you should remember when dispensing your samples into the gel well: a. Do not stab the pipet tip into the bottom of the well. This will poke a hole in the well and your sample will run out of the well and be lost in the buffer. b. Be sure that the tip is placed into the very top of the well, just below buffer level. If necessary, steady your hand on something (the lab bench, your other hand, etc.) so that your sample will fall into the well. Do NOT lean the pipet tip against the side of the well while dispensing the sample. I have watched several lab groups rip the wells apart doing this. c. When dispensing the sample, press down on the pipettor plunger slowly and consistently. If you push the sample out too quickly, it will rebound out of the well and be lost in the buffer.

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PCR Amplification of Cheek Cell DNA d. After loading the sample into a well, remember to keep the pipettor plunger depressed until after your remove your tip slowly from the buffer. If you do not, you risk sucking your carefully loaded sample back into the pipet tip! e. Do not forget to record the order in which you load your samples! There is a graphic of an agarose gel in the data section of the lab to do this. 5. Fill the pipet with the first control sample. Place the tip over the top of one of the wells. The tip should be submerged in the buffer at this point. Holding the pipet steady, gently dispense the sample into the well. The glycerin in the sample will allow the sample to sink into the well. Do not place the pipet tip directly into the well or you will risk poking a hole in the side or bottom of the well, and your sample may leak out of the gel. A new pipet tip should be used for each sample. Each student should have a turn loading a well. 6. Load the following amounts of each sample into adjacent wells. Wait for the instructor to give you gel loading instructions and make sure you change tips between samples. Lane 1 2 3 4 5 6 7 8

Sample MMR (DNA size standards) Homozygous (+/+) PCR control Homozygous (-/-) PCR control Homozygous (+/-) PCR control Student 1 Student 2 Student 3 Student 4

Volume loaded 10 µL (whole sample) 10 µL (whole sample) 10 µL (whole sample) 10 µL (whole sample) 15 µL 15 µL 15 µL 15 µL

NOTE: Any group with five students should leave out the homozygous (-/-) PCR control so that they have enough room for all five student samples in the gel. The (+/+) and (+/-) PCR controls should provide you with enough information. 7. Secure the lid on the box. The lid will attach to the base and sit evenly in only one orientation: red to red and black to black. Connect the electrical leads to the power supply in the same manner: red to red and black to black. 8. Use of Power Supplies a. Small power supplies: Attach the black power cord to the back of the power supply and plug the supply into an electrical outlet. On the front of the supply, select “100V” with the small black switch. Connect the electrophoresis chamber to the front of the power supply, being sure to match the color of the leads, red-to-red and black-to-black. Switch the power supply on using the black rocker switch. If the current is running properly, there will be a curtain of bubbles rising from the negative (black) electrode at the bottom of the electrophoresis chamber.

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PCR Amplification of Cheek Cell DNA b. Large power supplies: Before connecting the power supply to an outlet, make sure the black rocker switch on the unit is set to “OFF”. Make sure the unit is set for voltage (red light next to “V”) and then use the arrow keys to raise the digital read out from 0 to 100. Press the running figure on the far right hand side of the front panel and the digital read out should quickly increase from 0 to 100 volts. To verify that is current running through your electrophoresis chamber, look for a curtain of bubbles rising from the negative electrode at the bottom of the chamber. 9. Run the unit for 40 minutes. After 5 minutes check your gel to make sure the samples are running through the gel in the right direction (toward the red electrode)! Remember, the dye that you can visualize is not stained DNA. It is used as a marker to follow the progress of the samples through the gel. Staining the Gel 1. After 40 minutes have passed, turn the voltage off and turn the black rocker switch off at the back of the power supply. Then disconnect the electrodes from the power supply and take the lid off of the electrophoresis chamber. CAREFULLY remove the gel and casting tray making sure the gel does not slide out of the casting tray (cover both ends with fingers because the gel can be very slippery and slide right out of the tray…splat! There goes a week of work in the garbage). It is recommended that you wear gloves for this phase of the lab, as the DNA dye will stain your fingers blue. 2. Mark your tray with tape that has your initials on it. Do not write on the tray because other lab sections need to use it. 3. For this type of staining, two groups will share one staining tray. Place the two agarose gels in the staining tray by gently sliding the gel off of the casting tray. 4. Locate the bottle containing 1X DNA Fast Blast Dye solution and pour just enough into the staining tray to cover the gels (~120 mL/staining tray). 5. Cover the gels with tinfoil, and let them stain overnight. If possible, store the gels in a refrigerator during the staining, as it keeps the DNA bands clear and sharp. No destaining will be necessary with this dye. 6. Gently pour the used DNA Fast Blast stain from the staining tray into a waste beaker. Be sure the gels do not slip out! Carefully remove your gel and place it on some plastic wrap. Place the gel on a light background (like white paper, light box or overhead projector). 7. Determine whether you are homozygous (+/+ or -/-) or heterozygous (+/-) for the Alu insertion. If the results are ambiguous, have your instructor help you interpret the gel bands.  

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DATA SHEET

Name ________________________ Period _______ Class ___________ Date ___________

Results of PCR Product Electrophoresis Figure 1. Graphic Representation of PCR Product Electrophoresis Draw a picture of what the stained agarose gel looks like. Use a ruler to mark the distance (in mm) the samples have traveled from the bottom of each well.

Record order of samples loaded here

Wells

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Table 1: Individual Sample Data from Agarose Gel Record the numerical data obtained in Fig. 1 in the following table.

Lane

Number of Bands

M

4 (maybe 5)

1000, 700, 500, 200, (100)

+/+

1

941

+/-

2

941, 641

-/-

1

641

Distance (mm)

Size (bp)

Student 1 Student 2 Student 3 Student 4

Table 2: Class Genotypes Homozygous (+/+)

Heterozygous (+/-)

Homozygous (-/-)

# of students

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QUESTIONS & ANALYSIS OF RESULTS 1. What is the biological molecule that needs to be extracted from the cell for the PCR reaction? What membrane(s) need to be broken to retrieve this molecule?

2. Define the term polymorphism. What type of polymorphism is a SINE?

3. You specifically looked at the Alu SINE located at the PV92 locus on Chromosome 16 in this experiment. What does is mean to say the SINE is dimorphic at this locus? If a person is heterozygous for the Alu SINE, what would you find on each chromosome at this locus (drawing a picture might help with this)?

4. Why are there nucleotides (dC, dA, dT, dG) in the master mix? What are the other components of the PCR reaction?

5. From which organism was Taq DNA polymerase isolated? What is unique to this enzyme that allows it to function in the PCR reaction?

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PCR Amplification of Cheek Cell DNA 6. Describe the three main steps of each cycle of PCR amplification and what reactions occur at each temperature.

7. What controls are run in this experiment? Why are they important?

8. Use Data Table 2 and the equations given below to determine the frequency of each allele in your class sample Fill in the table below with your class data. Remember, a class of 32 students (N) will have a total of 64 (2N) instances of each locus. Hardy-Weinberg equation: p2 + 2pq + q2 = 1, therefore: p = frequency of (+) allele = number of (+) alleles total number of alleles (both + and –) = 2(# of +/+ students) + (# of +/– students) total number of alleles (both + and –) = frequency of (+/+) students + ½ (frequency of (+/–) students

q = frequency of (–) allele = number of (–) alleles total number of alleles (both + and –) = 2 (# of –/– students) + (# of +/– students) total number of alleles (both + and –) = frequency of (–/–) students + ½ (frequency of (+/–) students

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Table of Class Allelic Frequencies Category

Number

Frequency

(+) alleles

p=

(-) alleles

q= Total alleles =

= 1.00

9. The following table contains data from a USA-wide random population study of genotype at the PV92 locus. Table of USA-wide Genotype at PV92 Locus Category Number 2,422 Homozygous (+/+) 5,528 Heterozygous (+/-) 2,050 Homozygous (-/-) Total alleles = 10,000 Calculate the allelic frequencies for the US data as you did for the class data and fill in the table below. Category

Number

Frequency

(+) alleles

p=

(-) alleles

q= Total alleles = 10, 000

= 1.00

How do the class allelic frequencies and the random US-wide allelic data compare? Would you expect them to match? What reasons can you think of to explain the differences or similarities?

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