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Using an Alu Insertion Polymorphism to Study Human Populations6chromosomes, thus creating two possible alleles ( and –). These“dimorphic” Alus inserted within the last several hundred thousand years,reaching different allele frequencies in different human populations. Thus,Alu insertion polymorphisms are useful tools for reconstructing humanevolution and migration.KEY:Utah Pedigree 1356Female MaleCentre d'Etude du Polymorphisme Humain (CEPH)Genotyping by Renato Robledo / //NoData131331246512455Mendelian inheritance of the Aluinsertion ( ) at the PV92 21246312464124671246812469This experiment examines a human Alu dimorphism at the PV92 locus. Asample of human cells is obtained by saline mouthwash (alternativelyDNA may be isolated from hair sheaths). DNA is extracted by boiling withChelex resin, which binds contaminating metal ions. Polymerase chainreaction (PCR) is then used to amplify a chromosome region that containsthe PV92 Alu dimorphism. The Alu insertion allele ( ) is 300 nucleotideslonger than the non-insertion allele (–), so the two alleles are readilyseparated by agarose gel electrophoresis.Each student scores his or her genotype, and the compiled class resultsare used as a case study in human population genetics. Tools for testingHardy-Weinberg equilibrium, comparing the PV92 insertion in worldpopulations, and simulating the inheritance of a new Alu insertion arefound on the included CD-ROM or at the BioServers Internet site of theDolan DNA Learning Center (www.BioServers.org).Batzer, M.A., Stoneking, M., Alegria-Hartman, M., Barzan, H., Kass, D.H., Shaikh, T.H., Novick,G.E., Iannou, P.A., Scheer, W.D., Herrera, R.J., and Deininger, P.L. (1994). African Origin ofHuman-specific Polymorphic Alu Insertions. Proceedings of the National Academy ofSciences. USA 91: 12288-12292.Comas, D., Plaza, S., Calafell, F., Sajantila, A., and Bertranpetit, J. (2001). Recent Insertion ofan Alu Element Within a Polymorphic Human-specific Alu Insertion. Molecular Biologyand Evolution 18: 85-88.Deininger, P.L. and Batzer, M.A. (1999). Alu Repeats and Human Disease. Molecular Geneticsand Metabolism 67(3): 183-193.Mullis, K. (1990). The Unusual Origin of the Polymerase Chain Reaction. Scientific American262(4): 56-65.Prak, E.T.L. and Kazazian, H.H. (2000). Mobile Elements and the Human Genome. NatureReviews Genetics 1(2): 134-144.DNAKITSLearning CenterCopyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

Using an Alu Insertion Polymorphism to Study Human Populations7LAB FLOWI.ISOLATE DNA FROM CHEEK CELLS99 C(ALTERNATE) I. ISOLATE DNA FROM HAIR SHEATHS37 C99 CII. AMPLIFY DNA BY PCRIII. ANALYZE PCR PRODUCTS BY GEL ELECTROPHORESISCopyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

Using an Alu Insertion Polymorphism to Study Human Populations8METHODSI.ISOLATE DNA FROM CHEEK CELLSReagentsSupplies and Equipment0.9% Saline solution, 10 mL10% Chelex , 100 µL (in 0.2- or 0.5-mL PCRtube)Permanent markerPaper cupMicropipets and tips (10–1000 µL)1.5-mL microcentrifuge tubesMicrocentrifuge tube rackMicrocentrifuge adaptersMicrocentrifugeThermal cycler (or water bath or heatblock)Container with cracked or crushed iceVortexer (optional)1. Use a permanent marker to label a 1.5-mL tube and paper cup withyour assigned number.2. Pour saline solution into your mouth, and vigorously rinse your cheekpockets for 30 seconds.3. Expel saline solution into the paper cup.4. Swirl cup gently to mix cells that may have settled to the bottom. Usemicropipet with fresh tip to transfer 1500 µL of the solution into yourlabeled 1.5-mL microcentrifuge tube.5. Place your sample tube, along with other student samples, in abalanced configuration in a microcentrifuge, and spin for 90 secondsat full speed.Before pouring off supernatant,check to see that pellet is firmlyattached to tube. If pellet is looseor unconsolidated, carefully usemicropipet to remove as muchsaline solution as possible.6. Carefully pour off supernatant into the paper cup. Try to remove mostof the supernatant, but be careful not to disturb cell pellet at thebottom of the tube. (The remaining volume will approximately reachthe 0.1 mark of a graduated tube.)Food particles will not resuspend.7. Set micropipet to 30 µL. Resuspend cells in the remaining saline bypipetting in and out. Work carefully to minimize bubbles.Alternatively, you may add the cellsuspension to Chelex in a 1.5-mLtube, and incubate in a boilingwater bath or heat block.8. Withdraw 30 µL of cell suspension, and add to a PCR tubecontaining 100 µL of Chelex . Label the cap and side of the tubewith your assigned number.Your teacher may instruct you tocollect a sample of cell suspension toobserve under a microscope.9. Place your PCR tube, along with other student samples, in a thermalcycler that has been programmed for one cycle of the followingprofile. The profile may be linked to a 4 C hold program.Boiling step:The near-boiling temperature lysesthe cell and nuclear membranes,releasing DNA and other cellcontents.DNAKITSLearning Center99 C10 minutes10. After boiling, vigorously shake the PCR tube for 5 seconds.Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

Using an Alu Insertion Polymorphism to Study Human PopulationsTo use adapters, “nest” the sampletube within sequentially largertubes: 0.2 mL within 0.5 mL within1.5 mL. Remove caps from tubesused as adapters.911. Place your tube, along with other student samples, in a balancedconfiguration in a microcentrifuge, and spin for 90 seconds at fullspeed. If your sample is in a PCR tube, one or two adapters will beneeded to spin the tube in a microcentrifuge designed for 1.5-mL tubes.12. Use a micropipet with fresh tip to transfer 30 µL of the clearsupernatant into a clean 1.5-mL tube. Be careful to avoid pipettingany cell debris and Chelex beads.13. Label the cap and side of the tube with your assigned number. Thissample will be used for setting up one or more PCR reactions.14. Store your sample on ice or at –20 C until you are ready to continuewith Part II.I. (ALTERNATE) ISOLATE DNA FROM HAIR SHEATHSYour teacher may instruct you toprepare a hair sheath to observeunder a microscope.HAIR WITHSHEATHHAIRROOTBROKENHAIRReagentSupplies and Equipment100 mg/mL proteinase K, 100 µL (in 0.2or 0.5-mL tube)Permanent markerScalpel or razor bladeForceps or tweezersThermal cycler (or water bath or heatblock)Container with cracked or crushed iceVortexer (optional)1. Pull out several hairs and inspect for presence of a sheath. The sheathis a barrel-shaped structure surrounding the base of the hair, and canbe readily observed with a hand lens or dissecting microscope. Theglistening sheath can be observed with the naked eyes by holdingthe hair up to a light source. (Sheaths are most easily observed ondark hair.)2. Select one to several hairs with good sheaths. Alternately, select hairswith the largest roots. Broken hairs, without roots or sheaths, will notyield enough DNA for amplification.3. Use a fresh razor blade or scalpel to cut off hair shafts just above thesheath.4. Use forceps to transfer hairs to a PCR tube containing 100 µL ofproteinase K. Make sure sheath is submerged in the solution and notstuck on the test tube wall. Label the cap and side of the tube withyour assigned number.Alternatively, you may add thehairs to proteinase K in a 1.5-mLtube, and incubate in a water bathor heat block.5. Place your PCR tube, along with other student samples, in a thermalcycler that has been programmed for one cycle of the followingprofile.Incubation Step:37 C10 minutes6. Remove sample tube to room temperature. Vortex by machine orvigorously with finger for 15 seconds to dislodge cells from hair shaft.Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

Using an Alu Insertion Polymorphism to Study Human Populations107. Place your PCR tube, along with other student samples, in a thermalcycler that has been programmed for one cycle of the followingprofile. The profile may be linked to a 4 C hold program.Boiling step:99 C10 minutes8. Remove sample tube to room temperature, and mix by pipetting inand out for 15 seconds.9. Store your sample on ice or in the freezer until ready to begin Part II.II. AMPLIFY DNA BY PCRReagents (at each student station)Supplies and Equipment*Cheek cell or hair sheath DNA 2.5 µL(from Part I)*PV92B primer/loading dye mix, 25 µLReady-To-GoTM PCR beads (in 0.2-mL or0.5-mL PCR tube)Permanent markerMicropipet and tips (1-100 µL)Microcentrifuge tube rackThermal cyclerContainer with cracked or crushed iceShared ReagentMineral oil, 5 mL (depending on thermalcycler)*Store on ice1. Obtain a PCR tube containing a Ready-To-Go PCR Bead. Label withyour assigned number.The primer/loading dye mix will turnpurple as the PCR bead dissolves.2. Use a micropipet with fresh tip to add 22.5 µL of PV92B primer/loadingdye mix to the tube. Allow the bead to dissolve for a minute or so.If the reagents become splatteredon the wall of the tube, pool themby pulsing in a microcentrifuge orby sharply tapping the tubebottom on the lab bench.3. Use a micropipet with fresh tip to add 2.5 µL of your DNA (from Part I)directly into the primer/loading dye mix. Insure that no cheek cellDNA remains in the tip after pipetting.If your thermal cycler does nothave a heated lid: Prior to thermalcycling, you must add a drop ofmineral oil on top of your PCRreaction. Be careful not to touchthe dropper tip to the tube orreaction, or the oil will becontaminated with your sample.4. Store your sample on ice until your class is ready to begin thermal cycling.5. Place your PCR tube, along with other student samples, in a thermalcycler that has been programmed for 30 cycles of the followingprofile. The profile may be linked to a 4 C hold program after the 30cycles are completed.Denaturing step:Annealing step:Extending step:94 C68 C72 C30 seconds30 seconds30 seconds6. After cycling, store the amplified DNA on ice or at –20 C until you areready to continue with Part III.DNAKITSLearning CenterCopyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

Using an Alu Insertion Polymorphism to Study Human Populations11III. ANALYZE PCR PRODUCTS BY GEL ELECTROPHORESISReagentsSupplies and Equipment*PCR product (from Part II), 25 µLMicropipet and tips (1–100 µL)Microcentrifuge tube rackGel electrophoresis chamberPower supplyStaining traysLatex glovesUV transilluminator (for use with ethidiumbromide)White light transilluminator (for use withCarolinaBLU )Digital or instant camera (optional)Water bath (60 C)Container with cracked or crushed iceShared Reagents*pBR322/BstNI marker1.5% agarose in 1 TBE, 50 mL1 TBE, 300 mLEthidium bromide (1 µg/mL), 250 mLorCarolinaBLU Gel & Buffer Stain, 7 mLCarolinaBLU Final Stain, 250 mL*Store on ice1. Seal the ends of the gel-casting tray with masking tape, and insert awell-forming comb.Avoid pouring an overly thick gel,which is more difficult to visualize.The gel will become cloudy as itsolidifies.2. Pour 1.5% agarose solution to a depth that covers about 1/3 theheight of the open teeth of the comb.3. Allow the gel to solidify completely. This takes approximately20 minutes.4. Place the gel into the electrophoresis chamber, and add enough 1 TBE buffer to cover the surface of the gel.Do not add more buffer thannecessary. Too much buffer abovethe gel channels electrical currentover the gel, increasing runningtime.5. Carefully remove the comb, and add additional 1 TBE buffer to justcover and fill in wells, creating a smooth buffer surface.100-bp ladder may also be used asa marker.6. Use a micropipet with a fresh tip to load 20 µL of pBR322/BstNI sizemarker into the far left lane of the gel.Expel any air from the tip beforeloading. Be careful not to push thetip of the pipet through thebottom of the sample well.7. Use a micropipet with a fresh tip to add 25 µL of your sample/loadingdye mixture into your assigned lane of a 1.5% agarose gel, accordingto the diagram below. (If you used mineral oil during PCR, pierce yourpipet tip through the layer of mineral oil to withdraw the PCR sampleand leave the mineral oil behind in the original tube.)MARKERpBR322/BstNI12STUDENT SAMPLES34568. Run the gel at 130 V for approximately 30 minutes. Adequateseparation will have occurred when the cresol red dye front hasmoved at least 50 mm from the wells.Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

12Destaining the gel for 5–10minutes in tap water leachesunbound ethidium bromide fromthe gel, decreasing backgroundand increasing contrast of thestained DNA.Using an Alu Insertion Polymorphism to Study Human Populations9. Stain the gel using ethidium bromide or CarolinaBLU :a. For ethidium bromide, stain 10-15 minutes. Decant stain back intostorage container for reuse, and rinse gel in tap water. Use gloveswhen handling ethidium bromide solution and stained gels oranything that has ethidium bromide on it. Ethidium bromide isa known mutagen and care should be taken when using anddisposing of it.b. For CarolinaBLU , follow directions in the Instructor Planningsection.Transillumination, where the lightsource is below the gel, increasesbrightness and contrast.DNAKITSLearning Center10. View gel using transillumination, and photograph using a digital orinstant camera.Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

Using an Alu Insertion Polymorphism to Study Human Populations13BIOINFORMATICSFor a better understanding of the experiment, do the following bioinformaticsexercises before you analyze your results.Biological information is encoded in the nucleotide sequence of DNA.Bioinformatics is the field that identifies biological information in DNAusing computer-based tools. Some bioinformatics algorithms aid theidentification of genes, promoters, and other functional elements of DNA.Other algorithms help determine the evolutionary relationships betweenDNA sequences.Because of the large number of tools and DNA sequences available on theInternet, experiments done in silico (“in silicon,” or on the computer) nowcomplement experiments done in vitro (in glass, or test tube). Thismovement between biochemistry and computation is a key feature ofmodern biological research.In Part I you will use the Basic Local Alignment Search Tool (BLAST) toidentify sequences in biological databases and to make predictions aboutthe outcome of your experiments. In Part II you will identify additionalalleles at the PV92 locus. In Part III you will discover the chromosomelocation of the PV92 insertion.NOTE: The links in these bioinformatics exercises were correct at the timeof printing. However, links and labels within the NCBI Internet site changeoccasionally. When this occurs, you can find updated exercises athttp://bioinformatics.dnalc.org.I. Use BLAST to Find DNA Sequences in Databases (Electronic PCR)The following primer set was used in the experiment:5'-GGATCTCAGGGTGGGTGGCAATGCT-3' (Forward Primer)5'-GAAAGGCAAGCTACCAGAAGCCCCAA-3' (Reverse Primer)1. Initiate a BLAST search.a. Open the Internet site of the National Center for BiotechnologyInformation (NCBI) www.ncbi.nlm.nih.gov/.b. Click on BLAST in the top speed bar.c. Click on the link nucleotide BLAST under the heading Basic BLAST.d. Enter the sequences of the primers into the Search window. Theseare the query sequences.e. Omit any non-nucleotide characters from the window, becausethey will not be recognized by the BLAST algorithm.f. Under Choose Search Set, select the Nucleotide collection (nr/nt)database from the drop-down menu.Copyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

14Using an Alu Insertion Polymorphism to Study Human Populationsg. Under Program Selection, optimize for somewhat similar sequencesby selecting blastn.h. Click on BLAST! and the query sequences are sent to a server at theNational Center for Biotechnology Information in Bethesda,Maryland. There, the BLAST algorithm will attempt to match theprimer sequences to the millions of DNA sequences stored in itsdatabase. While searching, a page showing the status of yoursearch will be displayed until your results are available. This maytake only a few seconds, or more than a minute if a lot of othersearches are queued at the server.2. The results of the BLAST search are displayed in three ways as youscroll down the page:a. First, a graphical overview illustrates how significant matches, orhits, align with the query sequence. Matches of differing lengthsare coded by color. What do you notice?b. This is followed by a list of significant alignments, or hits, withAccession information.c. Next, is a detailed view of each primer sequence (query) aligned to thenucleotide sequence of the search hit (subject). Notice that a match tothe forward primer (nucleotides 1–25), and a match to the reverseprimer (nucleotides 26–51) are within the same Accession.3. What is the predicted length of the product that the primer set wouldamplify in a PCR reaction (in vitro)?a. In the list of significant alignments, notice the scores in the E-valuecolumn on the right. The Expectation or E-value is the number ofalignments with the query sequence that would be expected tooccur by chance in the database. The lower the E-value the higherthe probability that the hit is related to the query.b. Note the names of any significant alignments that have E-valuesless than 0.1. Do they make sense?c. Scroll down to the Alignments section to see exactly where the twoprimers have landed in this subject sequence.d. The lowest and highest nucleotide positions in the subjectsequence indicate the borders of the amplified sequence.Subtracting one from the other gives the difference between thetwo coordinates.e. However, the actual length of the fragment includes both ends, soadd 1 nucleotide to the result to determine the exact length of thePCR product amplified by the two primers.f. Is this the or the – allele?4. Now, take a closer look at this database hit, and copy its sequence forfuture use.a. Click on the Accession link at the left to open the sequencedatasheet for this hit.DNAKITSLearning CenterCopyright 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

Using an Alu Insertion Polymorphism to Study Human Populations15b. At the top of the report, note basic information about thesequence, including its basepair length, database accessionnumber, source, and references.c. The bottom section of the report lists the entire nucleotidesequence of the gene or DNA sequence that contains the PCRproduct. Highlight all the nucleotides between the beginning ofthe forward primer and end of reverse primer. Paste this sequenceinto a text document. Then, trim any extra nucleotides from theends, and delete all non-nucleotide characters and spaces. This isthe amplicon, or amplified product.II. Use BLAST to Identify Additional Alleles at the PV92 Locus1. Return to the nucleotide BLAST page.2. Paste the 416-bp PV92 amplicon, from 4.c. above, into the searchwindow. Ensure that Nucleotide collection (nr/nt) and blastn a

Boiling water bath (optional, see instructions) *Ready-to-Go PCR Beads incorporate Taq polymerase, dNTPs, and MgCl2. Each bead is supplied in an individual 0.5-mL tube or a 0.2-mL tube. **Electrophoresis reagents must be purchased separately for Kits 21-1230 and 21-1230A. Included