BIOTECH 2B03 Lab 7.docx

Experiment 7 – DNA Profile Analysis Using PCR Amplification and Agarose Gel Electrophoresis Introduction Although DNA from various individuals is more alike (Homo sapiens is 99.999% similar in its genetic makeup) than different, many regions of the human genome exhibit a great deal of diversity. Whe...

Experiment 7 – DNA Profile Analysis Using PCR Amplification and Agarose Gel Electrophoresis Introduction Although DNA from various individuals is more alike (Homo sapiens is 99.999% similar in its genetic makeup) than different, many regions of the human genome exhibit a great deal of diversity. When you multiple that 0.001% against the number of bases within the human genome (3,000,000,000), there are, on average, up to 30,000 genetic differences between any two individuals. These differences are termed “polymorphic”. The majority of these differences lie in the noncoding regions of the genome (i.e. pseudogenes, introns, promoters, nongene regions); however, some changes lie within the coding regions of genes. DNA identification methods use the DNA that is unique to generate a unique fingerprint. DNA fingerprinting or profiling uses recombinant DNA technology and has many applications, including diagnosing of genetic disease, forensic identification, paternity testing and criminal forensics. DNA profile analysis focuses on hyper variable regions of DNA. A short tandem repeat (STR) or Variable Number of Tandem Repeats segment (VNTR) is one such form of variability or polymorphism. These are composed of repeated copies of a DNA sequence that lie next to one another on the chromosome. One such genetic locus (D1S58) in the non-coding region of human chromosome 1, contains a VNTR sequence designated as pMCT118, which has a 16 bp consensus sequence. Most individuals have between 14 and 40 copies of the repeat on each of their copies of chromosome 1. The different versions of the pMCT118 polymorphism are referred to as alleles and are inherited in a Mendelian fashion on their maternal and paternal copies of chromosome 1. The VNTR locus has more than 29 different alleles and most people are heterozygous. This sort of variability allows for DNA profile analysis for paternity testing, and forensic evidence evaluation used during criminal investigations. The FBI uses a standard set of 13 STRs for CODIS (COmbined DNA Index System) analysis and databanking. In order to determine the number of repeats in an individual‘s mMCT118 alleles, primers are used that bracket the locus and result in selective amplification of that region of chromosome 1. The PCR products are separated by size by agarose gel electrophoresis. Each student‘s lane will have one or two bands visible, indicating whether they are homozygous or heterozygous at the pMCT118 locus. A band‘s position in the gel indicates the size and, hence, number of repeat units, of the pMCT118 allele. Figure 1 Maternal D1S58 DNA 5‘ 3‘ 3' 5' Primer 1 Primer 2 Paternal D1S58 DNA 5‘ 3‘ 3‘ 5‘ Another type of polymorphism is a Single Nucleotide Polymorphism (SNP). A single nucleotide is either changed to another nucleotide, deleted or added. Although there are 4 possible nucleotides, these polymorphism are usually biallelic i.e. they are found in only two forms. One way of detecting the presence of a SNP is if the change destroys or creates a Restriction Enzyme recognition sequence. Restriction Fragment Length Polymorphisms (RFLPs) are regions in the genome which vary between individuals that are detectable through the use of a specific restriction enzyme. A restriction enzyme is a specialized DNA cutting enzyme which only cuts the DNA when there is a specific sequence present (i.e. the enzyme EcoR1 only cuts within the sequence GAATTC). RFLPs occur when a random mutation creates or destroys one of these sites within a sequence. Example: Human DNA Sequence ATGGAATCCGCG (no EcoR1 RFLP absent) Variant Human Sequence ATGGAATTCGVG (EcoR1 RFLP present) Polymorphisms are sometimes within or linked to genes with a recognizable phenotype. One example of this is in the gene for the phenylthiocarbamide (PTC) taste receptor, TAS2R38. Variation in the ability to taste PTC is one of the most widely studied human genetic traits. The ability to taste PTC is an example of a simple Mendelian trait controlled by a dominant taster allele (T) and a recessive nontaster allele (t). (Actually, taster heterozygotes are slightly less sensitive to the taste of PTC when compared to taster homozygotes.) Nontasters (tt) know their genotypes since they have to be homozygous recessive to express their phenotype; however, tasters can be homozygous dominant or heterozygous (T-). It is now possible to use cleaved amplified polymorphic sequence (CAPS) analysis to determine genotypes for the PTC gene on the long arm of chromosome 7 (7q35-q36). Sequencing identified three nucleotide positions that vary within the human population – each a SNP. Two common alleles are found in most human populations. One specific combination of the three SNPs, termed a haplotype, correlates most strongly with tasting ability. (Analogous changes in other cell-surface molecules influence the activity of many drugs. For example, SNPs in the serotonin transporter and receptor genes predict adverse reactions to anti-depression drugs.) The nontaster allele produces a protein with alanine at amino acid position 49, valine at position 262, and isoleucine at position 296, and is referred to as the AVI allele. The common taster allele produces a protein with proline49, alanine262, and valine296, and is referred to as the PAV allele. Although the PAV and AVI alleles account for more than 90% of the variation in the PTC gene, a total of five additional alleles have been identified. One of the TAS2R38 SNPs occurs within the recognition sequence for the restriction enzyme HaeIII; one allele is cut by the enzyme and the other is not, resulting in a RFLP. Following electrophoretic separation of the digestion products, nontaster homozygotes will still see only the full length PCR product on the gel (221bp), while taster homozygotes will see two shorter fragments (177bp and 44bp) and taster heterozygotes will see all three fragment lengths. (Mammals are believed to distinguish only five basic tastes: sweet, sour, bitter, salty and umami (the taste of monosodium glutamate). Taste recognition is mediated by specialized taste cells that communicate with several brain regions through direct connections to sensory neurons. Taste perception is a two-step process: first, a taste molecule binds to a specific receptor on the surface of a taste cell; then the taste cell generates a nervous impulse, which is interpreted by the brain. Recent research has shown that taste sensation is determined by the wiring of a taste cell to the cortex, rather than the type of molecule bound by a receptor. So if a bitter taste receptor is expressed on the surface of a “sweet cell”, a bitter molecule is perceived as tasting sweet. Bitter-tasting compounds are recognized by receptor proteins on the surface of taste cells. The genetic basis of taste was first shown at DuPont in the early 1930‘s when some PTC dust escaped into the air when one chemist was transferring it. He tasted nothing but a lab-mate complained about the bitter taste of the dust. Subsequent studies showed that the inability to taste PTC is a recessive trait that varies in the human population. In addition to the 23 pairs of chromosomes in the nucleus, each mitochondrion has several copies of its own genome, and there are several hundred to several thousand mitochondria per cell. This means that the mitochondrial (mt) genome is highly amplified. Because of this high copy number, it is possible to obtain a mt DNA type from the equivalent of a single cell's worth of mt DNA. Thus, mt DNA is the genetic system of choice in cases where tissue samples are very old, very small, or badly degraded by heat and humidity. The mt genome contains 37 genes, all of which are involved in the production of energy and its storage in ATP. Genes take up the majority of the mt genome. However, there is a noncoding region of approximately 1,200 nucleotides spanning the arbitrary "0" position of the mt genome that is often referred to as the control region. This refers to the fact that this region contains the signals that control RNA and DNA synthesis. The DNA sequence of the control region accumulates point mutations at approximately 10 times the rate of nuclear DNA. The control region is relatively tolerant of a high mutation rate, because binding sites for DNA and RNA polymerase are defined by only short nucleotide sequences. The high mutation rate of mtDNA is likely due to the fact that the mt genome is located in close proximity to the respiratory machinery of the cell, a source of potent mutagens called oxygen free radicals. The mitochondrion and mt genome are inherited in a maternal lineage i.e. from mother to daughter or son. The relatively large egg has ~100,000 mitochondria; the tiny sperm has ~50-100 mitochondria, concentrated at the base of the tail, where they provide energy to power the flagellum. Very few male mitochondria are believed to enter the egg at the moment of conception, and those that do can easily be lost by "dilution" during mitosis or actively eliminated from the egg. The lack of paternal chromosomes simplifies the analysis of mitochondrial inheritance; the mt genome is inherited intact over thousands of generations, without the confounding effect of crossover with a paternal chromosome. Although new mutations occasionally arise, they are few and easy to discern. Because the mt genome is haploid - having only a maternal contribution - mt DNA types are termed haplotypes. This experiment examines a 1070-base pair sequence from the control region of mt genome. Because each student is amplifying the same region, the PCR products will be the same size for each. However, there is a polymorphic MseI site within this region. Digestion of the PCR product with the restriction enzyme will result in one of two possible cutting patterns: one for mtDNA lacking the MseI site and one for the mtDNA with the MseI site. The source of template DNA for all the procedures is a sample of several thousand squamous cells obtained from either hair sheaths or cheek cells. The root ends of hairs must be mixed with Chelex®/proteinase K to release the DNA; cheek cells only need to be mixed with Chelex®. For both, the samples are boiled to lyse the cells and liberate the DNA. The Chelex® binds metal ions that are released from the cells and that would otherwise inhibit the PCR reaction. The DNA-containing supernatant is then used in each of the PCR reactions with Taq polymerase, the four Deoxynucleotides (dNTPs), the cofactor MgCl2, and the appropriate primers. The mixture is placed in the thermal cycler and put through 30 – 35 cycles consisting of: a 30-second incubation at 94ºC, to denature the DNA a 30 or 40-second incubation at the appropriate temperature for specific primers to anneal a 30 or 45-second incubation at 72ºC, for the Taq polymerase to synthesize the DNA References Biotechnology, An Introduction. 2nd Edition. S. R. Barnum. 2005. Human VNTR Polymorphism AT. Carolina Biological Supply Company. Human Mitochondrial DNA Haplotyping Kit AT. Carolina Biological Supply Company, 2004 Merritt, R.B. et al. Tasting Phenylthiocarbamide (PTC): A New Integrative Genetics Lab with an Old Flavor. The American Biology Teacher, Online Publication, May 2008 Using a Single Nucleotide Polymorphism to Predict Bitter-Tasting Ability. Dolan DNA Learning Center, Cold Spring Harbor Laboratories, 2006. Preparation Questions Define the following terms: phenotype genotype polymorphism STR loci SNP loci RFLP single-locus allele haplotype upstream and downstream DNA primers If there are on average 30,000 genetic differences between any two people (due originally to mutations), why do we not all express more mutations? For forensic DNA profile analysis, is it an advantage, disadvantage, or neither to use a genetic locus in the non-coding region of the genome? Explain. Describe the purpose of the following reaction steps during each PCR cycle: Denaturation Annealing Extension Reagents 10% (w/v) Chelex® (10g Chelex® in 100ml H2O) Taq Polymerase with PCR buffer 10mM dNTP mix PCR primers 1 and 2 for pMCT118 containing loading dye (store on ice; freeze between use) PCR primers 1 and 2 for mtDNA containing loading dye (store on ice; freeze between use) PCR primers 1 and 2 for PTC containing loading dye (store on ice; freeze between use) 25mM MgCL2 Agarose (2% w/v) 1X TAE buffer GelRedTM 10,000X DNA size marker pBR322/BstN1 or 123 bp ladder (35 fragments) (store on ice; freeze between use) 0.9% NaCl solution (sterile) (0.9g NaCl/100ml distilled H2O MseI restriction enzyme (store on ice; freeze between use) HaeIII restriction enzyme (store on ice; freeze between use) BSA buffer (store on ice; freeze between use) Procedure Obtaining the DNA Template from Buccal (cheek) cells (Wear gloves for the entire procedure) Set up a boiling hot water bath tray. Use a permanent marker to place your assigned number on two clean 1.5-mL micro tubes and on a 15-mL test tube. Pour 10 mL saline (0.9% NaCl) solution into a clean paper cup. Rinse your mouth with drinking water first. Expel rinse water. Pour the saline solution into your mouth and vigorously rinse your mouth for 10 sec. Expel saline solution into the paper cup. Carefully pour saline solution from the paper cup into the 15 ml test tube and close cap tightly. Save paper cup for later use. Place your sample tube, in a balanced configuration in a large clinical centrifuge and spin for 10 min. Carefully pour off supernatant into the paper cup (Waste). Be careful not to disturb the cell pellet at the bottom of the test tube. Set micropipet to 500 µL. Draw 10% Chelex® suspension in and out of the pipet tip several times to suspend the resin beads. Before resin settles, rapidly transfer 500 µL of Chelex® suspension to the test tube containing your cell pellet. Re-suspend cells by pipetting in and out several times. Examine against light to confirm that no visible clumps of cells remain. Pipet several times to re-suspend cells and resin, then transfer 500 µL of your cell sample into a clean 1.5-mL microtube. Place your sample in a floating tube rack in the boiling water bath for 10 min. Do not submerge or drop the tube into the water. Use forceps to remove your tube from the boiling water bath and allow to cool for 2 min. Place your sample microtube, in a balanced configuration in the microcentrifuge and spin for 30 sec. Use a fresh tip to transfer 200 µL of the clear supernatant to a clean 1.5-mL tube. Be careful not to remove or disturb the Chelex®/cell debris at the bottom of the tube. Pour supernatant waste from Step 6 into the sink and rinse down with water. Obtaining the DNA Template from hair sheath cells (Wear gloves for the entire procedure) Pull out several hairs and inspect for presence of a sheath (a glistening, barrel-shaped structure surrounding the base of the hair). Select several hairs with good sheaths. Alternatively, select hairs with the largest roots (eyebrow hair is a good source of hair roots). Use a fresh razor blade or scalpel to cut off hair shafts just above the sheath. Use forceps to transfer hairs to a 1.5ml tube with 100μl of proteinase K/Chelex® mixture. Make sure the sheaths are submerged in the solution. Note: 10µl of Proteinase K is added to 200µl of 5% Chelex. Incubate the sample in a 50oC water bath for 10 minutes. Remove sample tube to room temperature. Vortex for 15 seconds to dislodge cells from hair shafts. Place your sample in a floating tube rack in a boiling water both for 8 minutes. Do not submerge or drop the tubes into the water. Use forceps to remove your tube and allow samples to cool for 2 minutes. The tube may be placed on ice for faster cooling. Vortex for 15 seconds. Label your tube and place it in a microfuge (with a balance if necessary) and spin at full speed for 30 seconds. Use a fresh tip to transfer 50μl of the clear supernatant to a clean 1.5ml tube. Be careful not to remove or disturb the Chelex®/cell debris at the bottom of the tube. Store sample on ice or freezer until you are ready to use it. Alternate DNA sources You can mimic forensic analysis by isolating DNA from cigarette butts. Cut three cross-sections, approximately 3 mm wide, from the filter end of a cigarette butt. Place the cuttings in a microfuge tube with 1 ml of 5% Chelex (1:2 dilution of stock) solution and vortex for 30 seconds. Then place the microfuge tube in a heating block at 56° C for 30 minutes, vortex, boil by heating to 100° C for eight minutes, vortex again, and centrifuge at 13,000 rpm for five minutes to pellet the Chelex. Experiment A: PCR Amplification of the pMCT118 VNTR Locus Use a micropipette with a fresh tip to add 22.5μl of pMCT118 primer/loading dye buffer mix to a 0.5ml PCR tube containing a Ready-To-Go PCR BeadTM. Tap tube with finger to dissolve bead until clear (wait a minute or two). Use a fresh tip to add 2.5μl of student DNA to the 0.5 ml PCR reaction tube, and tap to mix. (Insure that no DNA remains in the tip.) Pool reagents by pulsing in a microcentrifuge or by sharply tapping tube bottom on lab bench. Label the cap of your PCR tube with a name. Store all samples on ice, or in the freezer until ready to amplify, then run your samples in the PCR unit using the following profile. 94˚C for 30 sec. 65˚C for 30 sec. 72˚C for 30 sec. for 30 cycles Final cycle of 10 minutes at 72˚C Hold at 4˚C overnight (if required); label tube and freeze at -20˚C until ready for Electrophoresis. Experiment B: PCR Amplification of the mtDNA Use a micropipette with a fresh tip to add 22.5μl of mtDNA primer/loading dye buffer mix to a 0.5ml PCR tube containing a Ready-To-Go PCR BeadTM. Tap tube with finger to dissolve bead until clear (wait a minute or two). Use a fresh tip to add 2.5μl of student DNA to the 0.5 ml PCR reaction tube, and tap to mix. (Insure that no DNA remains in the tip.) Pool reagents by pulsing in a microcentrifuge or by sharply tapping tube bottom on lab bench. Label the cap of your PCR tube. Store all samples on ice, or in the freezer until ready to amplify, then run your samples in the PCR unit using the following profile: 94˚C for 30 sec. 58˚C for 40 sec. 72˚C for 45 sec. for 35 cycles Final cycle of 10 minutes at 72˚C Hold at 4˚C overnight (if required), label tube and freeze at -20˚C until ready for restriction digestion of PCR products and electrophoresis. Restriction digest of mtDNA PCR products (Week 2) 1. Use a micropipette with a fresh tip to add 15μl of each of your mtDNA PCR products to a fresh 1.5ml tube. Keep the remaining 10µl PCR product on ice for loading as undigested PCR product in step 10 of agarose gel electrophoresis. Use a fresh tip to add 2.25μl of 10x restriction buffer to the reaction tube. Use a fresh tip to add 2.25μl of 10x BSA buffer to the reaction tube. Use a fresh tip to add 9.5μl of sterile dH2O to the reaction tube. Use a fresh tip to add 1μl of MseI to the reaction tube. Pool the reagents by pulsing in the microcentrifuge or by sharply tapping the tube bottom on the lab bench. Label the cap of your tube and incubate the digest at 37ºC for 60 minutes. Use a fresh tip to add 3μl of 6x loading dye to your digest tube. Store on ice until ready for electrophoresis. Load the entire PCR digest sample/loading dye mixture. Experiment C: Using a SNP to predict bitter-tasting ability PCR Amplification of the PTC locus Use a micropipette with a fresh tip to add 22.5μl of PTC primer/loading dye buffer mix to a 0.5ml PCR tube containing a Ready-To-Go PCR Bead. Tap tube with finger to dissolve bead until clear (wait a minute or two). Use a fresh tip to add 2.5μl of student DNA to the 0.5 ml PCR reaction tube, and tap to mix. (Insure that no DNA remains in the tip.) Pool reagents by pulsing in a microcentrifuge or by sharply tapping tube bottom on lab bench. Label the cap of your PCR tube. Store all samples on ice, or in the freezer until ready to amplify, then run your samples in the PCR unit using the following profile: 94˚C for 30 sec. 64˚C for 45 sec. 72˚C for 45 sec. for 30 cycles Final cycle of 10 minutes at 72˚C Hold at 4˚C overnight (if required), label tube and freeze at -20˚C until ready for restriction digestion of PCR products and electrophoresis. Restriction digestion of PTC PCR products (Week 2) Use a micropipette with a fresh tip to add 15μl of your PTC PCR product to a fresh 1.5ml tube and label this tube D for “digested”. Keep the remaining PCR product (̴10µl) on ice for loading as “undigested” PCR product in step 10 of agarose gel electrophoresis. Use a fresh tip to add 1μl of restriction enzyme HaeIII into the tube labeled “D” Mix and pool the reagents by pulsing in the microcentrifuge or by sharply tapping the tube bottom on the lab bench. Label the cap of your tube and incubate the digest at 37ºC for 30 minutes. (This can be done in a thermal cycler programmed for one cycle at 37ºC for 30 minutes.) Store on ice until ready for electrophoresis. Load 16μl of the digested sample/loading dye mixture. Determining your PTC phenotype (Week 2) Place one strip of control taste paper in the centre of your tongue for several seconds. Note the taste. Remove the control taste paper, and place one strip of PTC taste paper in the centre of your tongue for several seconds. How would you describe the taste of the PTC paper as compared to the control: strongly bitter, weakly bitter, or no taste other than paper? DNA Analysis using Agarose Gel Electrophoresis: Week 2 **WEAR GLOVES WHEN HANDLING ETHIDIUM BROMIDE SOLUTION** Remove your PCR samples and DNA size marker from freezer. Weigh 1.5g of Agarose powder, and place in a 200ml beaker. Add 75ml of 1X TAE buffer. Microwave on medium for 1 to 1½ minutes until clear. Swirl and stir with a glass rod during the procedure. Remove any bubbles. When the beaker is cool enough to touch add 7.5μl of GelRed DNA stain (10,000X). Mix well. Assemble the gel electrophoresis equipment (see appendix if necessary). Pour the entire liquid gel onto the centre of the tray. Allow 20-30 minutes until gel is solid. Gently remove the comb from the casting tray. Position the gel and tray into the electrophoresis chamber with the wells towards the black electrode. Pour 1X TAE buffer into the ends of the casting tray. Fill to the mark. Do not overfill. For Experiment A, load entire volume of each of your PCR products using fresh tips and place in adjacent wells. For experiment B, load 10µl of each of your undigested PCR products and your entire PCR digested samples (or the maximum that fits in the well). For Experiment C, load 10μl of each of the undigested samples and 16μl of the digested samples. Note where each student sample is located into wells. Note: The cresol red and sucrose in the primer mix functions as a loading dye, so that undigested PCR products can be loaded directly into gels. Digested samples will need a loading dye added (6x). Load 10μl of the pBR322/BstN1 DNA ladder into a separate well. (Note which well.) Connect the lid and the power leads to the power supply. Set the power supply to 140 volts for 35 minutes. Run the gel. Turn off the power supply and remove the lid. Pour off the buffer. Remove the gel tray carefully and rinse the gel in distilled water. View and photograph gels using the gel doc system. Report Note: A condensed lab report is required for this lab that has the following components: Title page, Results and observations, Calculations, Post laboratory questions and references. You may hand in one report for per lab group. Make sure to reproduce one formal set of ‘Results/Observations’ in word-processed form. Also, include scans of all individual ‘Original Data’ with your name on the top of each scan you submit DNA samples move through the gel in a logarithmic fashion relative to the number of base pairs; i.e. the larger the fragment, the slower it moves. Using semi-logarithmic paper, you can plot the DNA ladder data and then compare your unknown samples to this.. DNA Ladder: Measure the distance in centimetres from the leading edge of the well, to the leading edge of each band in the DNA Ladder standard. Record this in Column A of Table 1. DNA Samples: Measure the distance in centimetres from the leading edge of the well to the leading edge of each band in the sample lanes. Record this in Column B of Table 1. Note: It is common to see an additional diffuse or fuzzy band lower on the gel. This is a “primer dimer”, an artifact of the PCR reaction that results from the primers overlapping one another and amplifying themselves. The primer dimer is approximately 40 - 50bp. Additional faint bands, at other positions, occur when the primers bind to chromosomal loci other than the specific one being tested, giving rise to “nonspecific” amplified products. Place the data in an approximate location to the size fragments of the DNA ladder. (The pBR322/BstN1 DNA Ladder marker contains bands at 1857, 1058, 929, 383 and 121 base pairs; the 100bp marker has multiples of 100bp) Preparing the Semi-logarithmic Graph: Divide the horizontal axis into cm units (1-7cm). Divide the logarithmic scale (vertical axis) into 100‘s and 1000‘s. Construct a standard plot using the DNA ladder data. Determine the number of base pairs for each sample fragment and record the values in Column C of Table 1. NOTE: For Experiment B, there are at least two haplotypes. The MseI+ haplotype is expected to have bands of 298bp, 288bp (not separable), 253bp, 161bp, 41bp and 38bp (not separable). The MseI-haplotype is expected to have bands of 541bp, 298bp, 161bp, 41bp and 38bp. (The 41bp and 38bp bands may not be visible on your gel.) Table 1: DNA Ladder/Unknown Fragment Size Comparison Student Unknown DNA DNA Ladder C B A DNA Ladder Fragment Estimated Fragment Migration Distance Migration Distance Size (bp) Size (bp) (cm) (cm) Post Laboratory Questions Experiment A How many bands are present in your DNA sample? Your lab partners DNA? Are you and your partners homozygous or heterozygous at the D1S58 locus? Explain. What would the presence of three differently sized bands resulting from one PCR reaction in this experiment suggest? Estimate the number of times the 16 bp core unit repeats for each band using your standard plot data. Assume that a PCR product containing one 16 bp repeat unit is 383 bp, a PCR product containing two 16 bp repeat units is 399 bp, etc. The sizes of the PCR products of the D1S80 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? Consider the following data from large populations: pMCT118 alleles: 29 pMCT118 heterozygosity: 72% of people pMCT118 matches: 1 in 18 people Do you think this protocol alone could be used to link a suspect with a crime or establish a paternity relationship? Why or why not? How could you modify the experiment to improve its ability to identify individuals? Experiment B From the results on your gel, do you appear to have the MseI+ or MseI- haplotype? Explain. How does this compare to the frequency of each Haplotype in the class? The mt control region mutates at approximately 10 times the rate of nuclear DNA. Propose a biological reason for the high mutation rate of mtDNA. How are mitochondrial restriction haplotypes limited in investigating genetic relationships and human evolution? (Hint: compare them to using nuclear genomic polymorphisms.) Experiment C Determine your PTC genotype. Locate the lane containing your unidigested PCR product (U). There should be one prominent band, and it should correspond to about 221bp. Compare your digested PCR product (D) with the uncut control. If you are homozygous recessive (tt) nontaster, you should see a single band in the same position as the uncut control. If you are homozygous dominant (TT) taster, you should see two bands of 177bp and 44bp. The 177bp band migrates just ahead of the uncut control; the 44bp band may be faint. (Incomplete digestion may leave a small amount of the uncut product, but this band should be clearly fainter than the 177bp band.) If you are a taster heterozygote (Tt), you should see three bands representing both alleles: 221bp, 177bp and 44bp. The 221bp band must be stronger than the 177bp band. (If it is fainter, it is an incomplete digestion of the PCR product.) Correlate your PTC genotype with your phenotype. How well did it predict your PTC-tasting ability? Research the terms synonymous and nonsynonymous mutation. Which sort of mutation are the polymorphisms in the TAS2R38 gene? The frequency of PTC nontasting is higher than would be expected if bitter-tasting ability were the only trait upon which natural selection had acted. It has been suggested that the PTC gene is under “balancing” selection, where a possible negative effect of losing this tasting ability is balanced by some positive effect. Under some circumstances, balancing selection can produce “heterozygote advantage”, where heterozygotes are fitter than either homozygote. What advantage might this be in the case of PTC? What ethical issues are raised by human DNA typing? Describe the function of the following reagents in these experiments: Be specific. Chelex 100 Taq Polymerase Proteinase K (used for Hair Sheath DNA Isolation) Discuss how cross contamination of samples may be problematic during the PCR process.