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1、stude nt Guide toDNA Fin gerpr intingby pcrExercise 1: Isolation of Cheek Cell DNAIn troduct ion:In this exercise, each stude nt will isolate DNA from his or her cheekcells (“ mouthwash DNA ); this DNA will later be amplified by the polymerase chairreaction (PCR). Cheek cells will first be collected

2、, by rinsing out your mouth with a saline solution (the salt keeps cells in proper osmotic balanee so they donThey are concentrated by spinning in a centrifuge, and then boiled with a resin (Chelex). During the boili ng, cells are disrupted and the DNA (now in sin gle- stra nded form) extracted in w

3、ater. The Chelex removes impurities that would otherwise in terfere with PCR.Equipme nt:Clinical centrifuge, micro centrifuge, 1000 bath with distilled water, floater.ypleh,pboetteswtaterrnal cSupplies:15ml coni cal cen trifuge tubes, paper cup, 1.5(two for each reaction), blue pipette tips, ice buc

4、ket, gloves and forceps.l micron trifuge tubesReage nts: 10ml of 0.9%(w/v) sali ne (NaCl) in distilled water; 10% Chelex suspe nsion (ion-excha nge resin).A note on cheek cells:Once cells have bee n collected into sali ne, it is importa ntto spin and concentrate them as soon as possible before they

5、start to burst. We will work with only 6 samples at a time, which fit into the clinical centrifuge. Once these are concen trated, we can start collect ing the n ext set of 6 samples.STEPINSTRUCTIONSVISUALSTEPONE: Tip:Use all three initials on sides and top of micro centrifuge tube.Write your in itia

6、ls CLEARLY on the 15ml cen trifuge tube containing sterile sali ne, on the paper cup, and on two 1.5& l micro centrifuge tubes(label tops ).Tip : KEEP ON ICE AT ALL TIMES!Sterile Saline15 ml centrifuge2-1.5 口 l micro centrifu ge tubesSTEPTWO:TipSlosh your mouth side to Side, very vigorously. Do not

7、eat, drink OR chew gum, preferably 1 hour before lab.STEPTHREEPour all of the sali ne soluti on into your mouth, slosh it around vigorously for 10 sec on ds, and expel it into the paper cup.Only 6 people can go at a time. Do not start sloshing until the centrifuge reads 1 minute. Slosh until you are

8、 ready to place your centrifuge tube directly into the centrifuge.Why saline?The salt keeps the cheek cells in proper osmotic balance so they don t burst. This is an isotonic state. IV s are .9 saline solutions also for proper osmotic balance.Pour the soluti on back into the 15ml cen trifuge tube, a

9、nd immediately place it into the clin ical cen trifuge.cup.STEPFOUR: Fact:This is called cell fractionation The reason for 10 minutes is to suspend the nuclei.Spin cells 10 minutes at maximum speed in the cen trifuge.STEPFIVETip:Try not to tip the tube back and forth; this will re-suspend the pellet

10、 and lose cells.Being careful not to disturb the pellet, pour the super nata nt into the paper cup. Try not to tip the tube back and forth; this will re-suspe nd the pellet and lose cells. Remove as much super nata nt as possible; whe n you notice the pellet starting to loose n stop pouri ng. (Addit

11、i onal super nata nt can be removed with a 1000 l pipette.)STEPSIXTip: Make sure they know to put a tip on theUsing a 1000口 l pipette set at 500pipette the Chelex suspe nsion up and dow n to re-suspe nd the beads. Immediately pipette 500口suspe nsion into your cheek cell pellet.pipette! When using a

12、micro pipette be careful not to turn over or under the maximum or minimum limits. Example: A 500 micro liter pipette should not be adjusted to 600.STEPSEVENTip: Do not invert the pipette tip! Make sure it is pointing down.What does Chelex do?The Chelex is made up of negatively charged microscopic be

13、ads that chelate or grab metal ions out of solution. It acts to trap metal ions, such as Mg+ which are required as catalysts or cofactors in enzymatic reactions. Your cheek cells will then be lysed or ruptured by heating to release all of their cellular components, including enzymes that were once c

14、ontained in the cheek cell Lysosomes. Lysosomes are sacs within the cells cytoplasm that contain powerful enzymes, such as DNAses which are used by clls to digest the DNA of invading viruses. When you rupture the cells, these DNAses can digest the released DNA of interest. However when the cells are

15、 lysed in the presence of the Chelex the cofactors are adsorbed and are not available to the enzymes. This blocks all enzyme degradation of the extracted DNA and results in a population of intact genomic DNA molecules that will be used as the template in your PCR reaction.Pipette up and dow n to mix

16、 res in and cells, and the n tran sfer cell pellet andres in to 1.5口 l micro cen trifuge tube.Check to make sure cap is secure.Make sure you disrupt the pellet by mixing the chelex and the pellet by pipetting up and down several times.STEPEIGHT:Tip:Watch closely with the forceps in case the top open

17、s due to pressure.Place tube in“ floater for 10 minu tes.Why water bath? This softens the plasma membranes and releases clumps of cells from each other. The increased temperature also acts to inactivate enzymes such as Dnases which will degrade the DNA template.ck and boilLTTTCTWater bath10 minu tes

18、.WATCH OUT FOR POPPING LIDS BE READY TO RESCUE THEM WITH FORCEPS! DO NOT LEAVESTEPNINETip:It is critical to get the tube on ice immediately!HQ1 mi nuteUNATTENDED!Remove tube from bath with forceps and place on ice for about 1 minuteWhy boil? The boiling ruptures the cells and releases the DNA from t

19、he cell nucleus. Your extracted genomic DNA will then be used as the target template for PCR amplification .PLACE ON ICE!STEPTENTip:Place the spine facing out in the centrifugeSTEP ELEVEN Tip:Remember the supernatant is the liquid on the top. That is your DNA! Do NOT get the pipette tip into the pel

20、let you do not want any Chelex in your tip.1 mi nuteSpin tubes in micro centrifuge (top speed) for 1 minute to pellet Chelex resin and impuritiesUsing 1000口 l pipette, transfer 200of supernatantto clean, labele d 1.5micro cen trifuge . Place on ice , or in freezer if not proceedi ng directly to PCR.

21、 This is your DNA sample. It will remain stable at -20 C for amon ths.NOW YOU ARE READY TO ADD YOUR DNA TO THE PCR MIX!92 (“ Alu ) PrimersPV92 Locus 5 Chromosame 16MAIERNALI_JRIGHTprime FtRESULTS OF GELelectrophoresis715 tp415 tpPftiWEhDIMERExperiment 2: PCR amplification with PV-The true power of P

22、CR is the ability to target and amplify a specific piece of DNA (or gene) out of a complete genome that con sist of 30,000 gen es.In troduct ion:This experime nt exam ines PV92, a huma n-specificAlu in serti on onchromosome 16. The PV92 gen etic system has only two alleles in dicat ing the prese nee

23、 (+) or abse nee (-) of the Alu tran sposable eleme nt on each of the paired chromosomes. This results in three PV92 geno types (+, +-, or -). The + and - alleles can be separated by size using gel electrophoresis. The part of your DNA that actually codes for any thi ng is only about 5% of your tota

24、l chromosomal DNA or geno me. The rema ining 95% con sists of stretches betwee n gen es, and in terrupt ing seque nces withi n genes (intron s). Much of this non-codi ng DNA is thought to be“ junk ，in that it does n t affect phe no type. For in sta nee, our chromosomes haveapproximately 500,000 copi

25、es of a 300 base-pair sequenee (called an“ Alu sequenee). This junk“ Alu DNA actually makes up abou05%of genomic DNA-as much as all out genes put together! (The presenee of“ Alu sequerchromosomes is thanks to an ancient retrovirus in which once infected our ancestors. This virus, a distant relative

26、of the AIDS virus, copied cellular RNA sequences into DNA and stuck then in at random chromosomal locations). Since it located withi n a non-codi ng porti on of a gen e, it does n t affect gene expressi on.This particular“ Alu insertioiaveenapptenled in the past million years, in arece nt huma n an

27、cestor. As a result, some huma n chromosomes have it and others don t. It is stably in herited accord ing to Men del s rules. At this particular glocus (PV-92), there are two alleles: Alu-prese nt and Alu-abse nt. They can be detected by amplifying the locus with primers flanking the Alu insertion.

28、After PCR amplification, the Alu-present allele gives rise to a 715 bp fragment; the Alu-absent allele yields a 415 bp fragme nt. These can be separated by gel electrophoresis in agarose. People can be homozygous for the Alu-prese nt allele (one 715 bp fragme nt); heterozygous (one 715 bp and one 41

29、5 bp band) or homozygous for the Alu-abse nt allele (one 415 bp ban d).Equipme nt: Cycler, micro cen trifuge, 100ul pipette. Supplies: Yellow pipette tips, gloves, two cycler tubes. Reage nts: (in ice bucket)Your cheek cell DNAAlu PCR mix (already in cycler tube)Note to teacher:Pre- warm thermal cyc

30、ler by running program-soak mobovpSTEPONE:Tip:Use all three initials on sides and top of micro centrifuge tube.Tip two:The heat from your hands on the bottom of your tube can ruin your DNA. That s becaus your HOT!Hold the tube near the top gently.? Get a PCR tube from your teacher.? Label your PCR t

31、ube with your in itials.? The PCR tube will co ntain 27 micro-liters of PCR mix that your teacher has prepared for you.? These tubes are fragile, and should be han dled only in the special racks provided.? If you take one out and squeeze it slightly it will crack and leak!? When you HAVE to remove i

32、t from a rack, han dle it gen tly n ear the top.The base is the most fragile part.Tip: KEEP ON ICE AT ALL TIMES!What is in the PCR mix?n1. DNA template (Your DNA)2. Individual deoxynucleotide bases (A,T,C,G)3. TAQ polymerase4. Magnesium ions a cofactor (catalyst) required by DNA polymerase to create

33、 the DNA chain.5. Oligonucleotide primers pieces of DNA complementary to the template that tell DNA polymerase exactly where to star making copies.6. Salt buffer, which provides the optimum environment and pH for PCR reaction.The PCR machine or thermocycler will run through 30 cycles.What is TAQ pol

34、ymerase? This is Dna polymerase that has been isolated from a heat stable bacterium ( Thermus aquaticus ) Which in nature lives within the steam vents in the Yellowstone National Park. For this reason the enzymes within these bacteria have evolved to withstand high temperatures (95 degrees C) also u

35、sed in each cycle of the PCR reaction.STEPTWO:Keep ice out of open tubes!Set up a react ions as follows: (on ice- be careful to keep ice out of ope n tubes) You are now going to add 3 micro-liters of DNA to the PCR mix that is in your PCR tube.When addi ng the3ul to your PCR mix place theDNA on the

36、side of the PCR tube so you can see it is in the tube. Show your teacher!keSTEPTHREE:STEPMix gen tly by placi ng in micro cen trifuge (use adaptors and balaneed configuration-teacher will dem on strate).Pulse briefly at maximum speed.SIGN UP FOR YOUR PLACE IN THE PCRCemlritugpFOUR:MACHINE. PLACE ALL

37、 3 INITIALS ON INNow you are ready to amplify your DNA!This is where you only need a little bit of DNA because the thermocycler amplifies it.THE SLOT.Place tube in rack, secure cap with cap tool.Place in thermal cyclerSTEPFIVE:mjDobp.PCR STEPS:Start programTip: Teacher will set this up.Look below to

38、 see what is happening inside the thermocycler or PCR machine.:Cli i i i r ir imr i:i r r r in?On血uw Strands 肺 IrC1rniniinrmAnneal! I. ；|- |*In our lab we repeated the cycle, 30 times.1. Denature Strands 95 C? The temperature is so hot the hydrogen bonds between the bases break and the two strands s

39、eparate.2. Hybridization 58 C? The primers anneal (bond) to the parental strands. Remember these primers are specific for the loci that is being amplified.3. DNA Synthesis 72 C? Taq polymerase loves this temperature and starts elongating the strands by adding the complementary bases.STEPSIX:At end o

40、f program, teacher will freeze your amplified DNA.Where are introns located? Introns are found betwee n exons. They are the non- codi ng segme nts. They are removed before tran slati on, in RNA process ing. Remember RNA process ing after tran scripti on? RNA process ing cuts out the introns so the e

41、xons or codi ng segme nt can be sent to the ribosome by messe nger RNA. Remember spliceosomes remove the introns. Alu is found in the introns.Below is a picture of what we are look ing at. Alu or PV92 is an intron located betwee n exons. It doesn t code for a protein so it does not show anything imp

42、ortant other than a link to an evolutio nary past.EXPERIMENT ONE: PRACTICE GELS USING CHROMATRACK DYESSTEP ONEPrepare TAE buffer by addi ng 100 ml of 10x TAE buffer to 900 ml super water already in the supplied bottle. Use the 1x TAE buffer for pour ing and running the gel. Chill rema ining 1x TAE b

43、uffer.(5 minu tes prep time)STEP TWOTip: This will be clear whe n ready.For each gel you prepare ADD .36 GRAMS OF REGULAR AGAROSE TO 30 mL OF 1x TAE buffer. Set up gel apparatus Boil and swirl the gel solution until it is completely dissolved in solution. (Use settings 1 and 2 in alter nati on, and

44、check freque ntly).STEP THREEAllow soluti on to cool to about 60 C beforepouri ng. This will be hot to the touch but not pain ful.STEP FOURPrepare gel tray by seali ng ends with tape or other custom-made dam. You can use mask ing tape or the rubber dams provided.STEP FIVETip:The comb has to bespaced

45、 away from the edge so the wells are not up against the edge. Make sure you use a spacer.Place comb in gel tray using spacer provided. Make sure you use spacer. Positi on the comb vertically such that the teeth are about 1-2 mm above the surface of the tray.STEP SIXTip:If air bubbles appearuse a dis

46、posable pipette to get the bubble out.STEP SEVENTip:When removing thecomb, make sure the buffer is covering the gel. This keeps the gel from tearing. Be very careful removing the rubber bumpers when using metaphor agarose. These gels will tear very easily.Pour 60 C gel solutioninto tray to a depth o

47、f about 5 mm. Allow gel to solidify about 20 minu tes at room temperature.Whe n gel is cooled (about 30 minu tes, it should be grayish), pour 1X TAE buffer over it to fill the buffer chambers and cover the gel to about 3 mm. Place the apparatus in the refrigerator and chill about 30 minu tes before

48、remov ing the comb and bumpers. Gen tly remove the comb, place tray in electrophoresis chamber, and cover (just un til wells are submerged) with electrophoresis buffer (the same buffer used to prepare the agarose) Load ing the gel: You have three dye soluti ons to run on practice gels. One is a dilu

49、ted soluti on of 5xTAE you no rmally use for PCR react ions. Ano ther is a soluti on containing the same dye we no rmally use, as well as a sec ond lighter blue dye, which runs more slowly. This solution is in the tube labeled BPB/XC. Fi nally you have chromatrack,Tip: Make sure the tip of the pipet

50、te is below the buffer yet above the floor of the well. Becauseof glycerol the tip just has to break the surfaceand the sample will end up in the right place. If you push the pipette tip toLoad 15 micro-liters, of each soluti on into lanes in whatever comb in ati on you like.s denRtyn the gel at 70

51、Volts remembering to set the gel up so that DNA runsto the positive end orred electrode!This will take 1 hour.which contains several dyes with multiple colors and separati on characteristics.deep it will pun cture the gel.WHAT SHOULDYOU SEE?DNA TAE AGAROSEDYE1.0%AGAROSEBLUE6,500 BpsYELLOW2,600 BpsRE

52、D1,500 BpsBLUE1,100 BpsFUCHSIA500 BpsORANGE100 BpsThis is depe ndent on the % agarose used. We use 1.0% so they migrate approximately equal to the chart show n to the left.Why does orange move so far away from the wells?An swer: the smaller fragme nts move the farthest away.Experime nt 3: Gel Electr

53、ophoresisSTEP ONE:Tip:A 123 base pair ladder will be added first to use as a reference.Tip two:Each student will write their number on the sheet of paper provided by the teacher so they know what lane their DNA is in. We will use numbers instead of initials. Record your number in your lab book so yo

54、u don etforgThe teacher will give you your DNA that has been amplified through PCR. This is in the freezer.Note: Lane 1 will be use for the 123 base pair ladder. We will use this to cou nt the base pairs like 415 and 715.STEP TWO:Tip:When pipeting make sure you steady your hand using your finger and

55、 go to the first stop, hold down releasing the DNA and take out of buffer with the plunger still down. Do not let go of the plunger until you are out of the TAE buffer Remember that you don want to pierce the gel with the pipette. The DNA will go into the well due to gravity.Stand in line and wait t

56、o load your gel. Please be respectful of others and don the table or play arou nd.Make sure you use a new sterile tip on your pipette.Each stude nt will add 15 micro-liters of their DNA to the well in the gel provided by the teacher.t ? Remember to write down your numberSTEP THREE:Does DNA run negat

57、ive to positive or positive to negative?Answer: Negative to positive because the dye and DNA is negatively charged. DNA has a phosphateWhe n every one is fini shed loadi ng their well, the teacher will turn on the power supply to 70 volts!This will run for 11/2 to 2 hoursbackbone that gives it a negative charge.Step FOUR:Safety:Goggles and gloves are TO BE WORN!Stai ningThe gel will be carefully placed into a well boat using a piece of film.STEP FIVE:Safety:Goggles and glove