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Student Guide to DNA Fingerprinting by PCR

By Tom Ray,2014-12-23 12:42
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Student Guide to DNA Fingerprinting by PCR

    Student Guide to DNA Fingerprinting by PCR

    Exercise 1: Isolation of Cheek Cell DNA

    Introduction: In this exercise, each student will isolate DNA from his or her cheek cells (“mouthwash DNA”); this DNA will later be amplified by the polymerase chain reaction (PCR). Cheek cells will first be collected, by rinsing out your mouth with a saline solution (the salt keeps cells in proper osmotic balance so they don’t burst). They are concentrated by spinning in a centrifuge, and then boiled with a resin (Chelex). During the boiling, cells are disrupted and the DNA (now in single-stranded form) extracted in water. The Chelex removes impurities that would otherwise interfere with PCR.

Equipment:

    Clinical centrifuge, micro centrifuge, 1000μl pipettes, thermal cycler, boiling water

    bath with distilled water, floater.

    Supplies: 15ml conical centrifuge tubes, paper cup, 1.5μl micro centrifuge tubes (two for each reaction), blue pipette tips, ice bucket, gloves and forceps.

    Reagents: 10ml of 0.9%(w/v) saline (NaCl) in distilled water; 10% Chelex suspension (ion-exchange resin).

    A note on cheek cells: Once cells have been collected into saline, it is important to spin and concentrate them as soon as possible before they start to burst. We will work with only 6 samples at a time, which fit into the clinical centrifuge. Once these are concentrated, we can start collecting the next set of 6 samples.

STEP INSTRUCTIONS VISUAL

    Write your initials CLEARLY on the STEP

    15ml centrifuge tube containing ONE:

    sterile saline, on the paper cup, and Tip:

    on two 1.5μl micro centrifuge tubes Use all three Sterile Saline 15 ml centrifuge initials on (label tops). sides and top of micro centrifuge Tip: KEEP ON ICE AT ALL TIMES!! 2- 1.5 μl micro centrifuge tubes tube.

Student Guide to DNA Fingerprinting by PCRb HELWIG 1

    Pour all of the saline solution into STEP

    your mouth, slosh it around vigorously TWO:

    for 10 seconds, and expel it into the Tip

     paper cup. Slosh your Crimp one side to make a spout on the Dixie mouth side to Only 6 people can go at a time. Do not start sloshing cup. Side, very until the centrifuge reads 1 minute. Slosh until you vigorously. Do are ready to place your centrifuge tube directly not eat, drink into the centrifuge.

    OR chew gum,

    preferably 1 Why saline?

    hour before The salt keeps the cheek cells in proper osmotic lab. balance so they don’t burst. This is an isotonic

    state. IV’s are .9 saline solutions also for proper

    osmotic balance.

    Pour the solution back into the 15ml STEP

    centrifuge tube, and immediately THREE

    place it into the clinical centrifuge.

    Spin cells 10 minutes at maximum STEP

    speed in the centrifuge. FOUR:

    Fact: This is called

    cell

    fractionation.

    The reason for

    10 minutes is

    to suspend the

    nuclei.

    Being careful not to disturb the STEP

     pellet, pour the supernatant into the FIVE

    Tip: paper cup. Try not to tip the tube Try not to tip back and forth; this will re-suspend the tube back the pellet and lose cells. Remove as and forth; this will re-suspend much supernatant as possible; when the pellet and you notice the pellet starting to lose cells.

    loosen stop pouring. (Additional

    supernatant can be removed with a

    1000μl pipette.)

    Using a 1000μl pipette set at 500μl, STEP

    pipette the Chelex suspension up and SIX

    down to re-suspend the beads.

    Immediately pipette 500μl of Chelex Tip: Make

    suspension into your cheek cell pellet. sure they

    know to put a tip on the Student Guide to DNA Fingerprinting by PCRb HELWIG 2

    pipette! What does Chelex do? When using a The Chelex is made up of negatively charged micro pipette microscopic beads that chelate or grab metal ions be careful not out of solution. It acts to trap metal ions, such as to turn over or Mg+ which are required as catalysts or cofactors in under the enzymatic reactions. Your cheek cells will then be maximum or lysed or ruptured by heating to release all of their minimum limits. cellular components, including enzymes that were Example: A once contained in the cheek cell Lysosomes. 500 micro liter Lysosomes are sacs within the cells cytoplasm that pipette should contain powerful enzymes, such as DNAses which not be are used by clls to digest the DNA of invading adjusted to viruses. When you rupture the cells, these DNAses 600. can digest the released DNA of interest. However when the cells are 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 down to mix resin and STEP

    cells, and then transfer cell pellet and SEVEN

    Tip: Do not resin to 1.5μl micro centrifuge tube. invert the Check to make sure cap is secure. pipette tip! Make sure it is

    pointing down. Make sure you disrupt the pellet by

    mixing the chelex and the pellet by

     pipetting up and down several times.

    Place tube in “floater” rack and boil STEP

    for 10 minutes. EIGHT:

     Tip:

    Watch closely Why water bath? This softens the plasma with the membranes and releases clumps of cells from each 10 minutes. forceps in case other. The increased temperature also acts to WATCH OUT FOR POPPING LIDS the top opens inactivate enzymes such as Dnases which will BE READY TO RESCUE THEM due to degrade the DNA template. WITH FORCEPS!! DO NOT LEAVE pressure. UNATTENDED!

    Remove tube from bath with forceps STEP

    and place on ice for about 1 minute. NINE Why boil? The boiling ruptures the cells and Tip: releases the DNA from the cell nucleus. Your It is critical to extracted genomic DNA will then be used as the 1 minute get the tube target template for PCR amplification. on ice immediately! PLACE ON ICE!!!!!

    Student Guide to DNA Fingerprinting by PCRb HELWIG 3

    Spin tubes in micro centrifuge (top STEP

    speed) for 1 minute to pellet Chelex TEN

    resin and impurities Tip: 1 minute

    Place the spine

    facing out in

    the centrifuge

    Using 1000μl pipette, transfer 200μl STEP

    of supernatant to clean, labeled 1.5μl ELEVEN

    micro centrifuge. Place on ice, or in Tip:

    freezer if not proceeding directly to Remember the

    supernatant is PCR. This is your DNA sample. It will the liquid on remain stable at -20?C for a few the top. That

    is your DNA! months. DNA Do NOT get the pipette tip

    NOW YOU ARE READY TO ADD into the pellet

    you do not YOUR DNA TO THE PCR MIX!! want any

    Chelex in your

    tip.

Student Guide to DNA Fingerprinting by PCRb HELWIG 4

    Experiment 2: PCR amplification with PV-92 (“Alu”) Primers

    The true power of PCR is the ability to target and amplify a specific piece of DNA (or gene) out of a complete genome that consist of ~30,000 genes.

Introduction: This experiment examines PV92, a human-specific Alu insertion on

    chromosome 16. The PV92 genetic system has only two alleles indicating the presence (+) or absence (-) of the Alu transposable element on each of the paired

    chromosomes. This results in three PV92 genotypes (++, +-, or --). The + and - alleles can be separated by size using gel electrophoresis. The part of your DNA that actually codes for anything is only about 5% of your total chromosomal DNA or genome. The remaining 95% consists of stretches between genes, and interrupting sequences within genes (introns). Much of this non-coding DNA is thought to be “junk”, in that it doesn’t affect phenotype. For instance, our chromosomes have approximately 500,000 copies of a 300 base-pair sequence (called an “Alu”

    sequence). This junk “Alu” DNA actually makes up about 5% -10% of genomic DNA-

    as much as all out genes put together! (The presence of “Alu” sequences in our chromosomes is thanks to an ancient retrovirus in which once infected our ancestors. This virus, a distant relative of the AIDS virus, copied cellular RNA sequences into DNA and stuck then in at random chromosomal locations). Since it’s located within a non-coding portion of a gene, it doesn’t affect gene expression. This particular “Alu” insertion seems to have happened in the past million years, in a

    recent human ancestor. As a result, some human chromosomes have it and others don’t. It is stably inherited according to Mendel’s rules. At this particular genetic locus (PV-92), there are two alleles: Alu-present and Alu-absent. They can be detected by amplifying the locus with primers flanking the Alu insertion. After PCR amplification, the Alu-present allele gives rise to a 715 bp fragment; the Alu-absent allele yields a 415 bp fragment. These can be separated by gel electrophoresis in agarose. People can be homozygous for the Alu-present allele (one 715 bp fragment); heterozygous (one 715 bp and one 415 bp band) or homozygous for the Alu-absent allele (one 415 bp band).

Student Guide to DNA Fingerprinting by PCRb HELWIG 5

    Equipment: Cycler, micro centrifuge, 100ul pipette. Supplies: Yellow pipette tips, gloves, two cycler tubes.

    Reagents: (in ice bucket)

    Your cheek cell DNA

    Alu PCR mix (already in cycler tube)

Note to teacher: Pre-warm thermal cycler by running program “mobovp-soak”

     ; Get a PCR tube from your teacher. STEP ; Label your PCR tube with your initials. ONE: ; The PCR tube will contain 27 micro-liters of Tip: PCR mix that your teacher has prepared for Use all three you. initials on ; These tubes are fragile, and should be handled sides and top of micro only in the special racks provided. centrifuge ; If you take one out and squeeze it slightly it tube. will crack and leak!

    Tip two: ; When you HAVE to remove it from a rack, The heat from handle it gently near the top. your hands on the bottom of your tube can The base is the most fragile part. ruin your DNA. The PCR machine or thermocycler That’s because will run through 30 cycles. Tip: KEEP ON ICE AT ALL TIMES!! your HOT! What is in the PCR mix? What is TAQ polymerase? Hold the tube This is Dna polymerase that has near the top been isolated from a heat stable gently. 1. DNA template (Your DNA) bacterium (Thermus aquaticus) 2. Individual deoxynucleotide bases (A,T,C,G) Which in nature lives within the 3. TAQ polymerase steam vents in the Yellowstone 4. Magnesium ions a cofactor (catalyst) required by National Park. For this reason the DNA polymerase to create the DNA chain. enzymes within these bacteria 5. Oligonucleotide primers pieces of DNA have evolved to withstand high complementary to the template that tell DNA temperatures (95 degrees C) also polymerase exactly where to star making copies. used in each cycle of the PCR 6. Salt buffer, which provides the optimum reaction. environment and pH for PCR reaction.

    STEP Set up a reactions as follows: (on ice- be

    TWO: careful to keep ice out of open tubes)

    Keep ice out You are now going to add 3 micro-liters of DNA of open to the PCR mix that is in your PCR tube. tubes! When adding the 3ul to your PCR mix place the DNA on the side of the PCR tube so you can see

     it is in the tube. Show your teacher!

    Student Guide to DNA Fingerprinting by PCRb HELWIG 6

STEP Mix gently by placing in micro centrifuge (use

    THREE: adaptors and balanced configuration-teacher

    will demonstrate).

    Pulse briefly at maximum speed.

    STEP SIGN UP FOR YOUR PLACE IN THE PCR FOUR: MACHINE. PLACE ALL 3 INITIALS ON IN

     THE SLOT.

    Now you are Place tube in rack, secure cap with cap tool. ready to Place in thermal cycler amplify your

    DNA!!

    This is where you only need

    a little bit of

    DNA

    because the

    thermocycler

    amplifies it.

    STEP Start program “mobobp-alu”.

    Tip: Teacher will set this up. FIVE:

    Look below to see what is happening inside the thermocycler

    or PCR machine. PCR STEPS: 1. Denature Strands 95? C ; The temperature is so hot the hydrogen bonds between the bases break and the two strands separate. 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. In our lab we repeated the cycle, 30 times.

    Student Guide to DNA Fingerprinting by PCRb HELWIG 7

STEP At end of program, teacher will freeze your

    SIX: amplified DNA.

    Where are introns located? Introns are found between exons. They are the non-coding segments. They are removed before translation, in RNA processing. Remember RNA processing after transcription? RNA processing cuts out the introns so the exons or coding segment can be sent to the ribosome by messenger RNA. Remember spliceosomes remove the introns. Alu is found in the introns.

Below is a picture of what we are looking at. Alu or PV92 is an intron located between

    exons. It doesn’t code for a protein so it does not show anything important other than a

    link to an evolutionary past.

Student Guide to DNA Fingerprinting by PCRb HELWIG 8

    EXPERIMENT ONE: PRACTICE GELS USING CHROMATRACK DYES

    Prepare TAE buffer by STEP ONE

    adding 100 ml of 10x TAE buffer to 900 ml super water already in the supplied bottle. Use the 1x TAE buffer for pouring and running the

    gel. Chill remaining 1x TAE buffer.

    (5 minutes prep time)

    For each gel you prepare STEP TWO

    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 Tip: This will be completely dissolved in

    clear when ready. solution. (Use settings 1 and 2 in alternation, and check frequently).

    Allow solution to cool to STEP THREE

    about 60?C before

    pouring. This will be hot to the touch but not painful.

    Prepare gel tray by sealing STEP FOUR

    ends with tape or other custom-made dam. You can use masking tape or the rubber dams provided.

    Place comb in gel tray STEP FIVE

    using spacer provided.

    Make sure you use spacer. Tip: The comb has to be

    Position the comb spaced away from the edge so

    the wells are not up against the vertically such that the edge. Make sure you use a teeth are about 1-2 mm spacer.

    above the surface of the tray.

    Student Guide to DNA Fingerprinting by PCRb HELWIG 9

Pour 60?C gel solution STEP SIX

    into tray to a depth of Tip: If air bubbles appear

    use a disposable pipette to get about 5 mm. Allow gel to the bubble out. solidify about 20

     minutes at room

    temperature.

    When gel is cooled (about STEP SEVEN

    30 minutes, it should be

    grayish), pour 1X TAE Tip: When removing the

    buffer over it to fill the comb, make sure the buffer

    is covering the gel. This buffer chambers and

    keeps the gel from tearing. cover the gel to about 3 Be very careful removing mm. Place the apparatus the rubber bumpers when in the refrigerator and using metaphor agarose.

    chill about 30 minutes These gels will tear very

    easily. before removing the comb and bumpers.

    Gently remove the comb, place tray in

    electrophoresis chamber, and cover (just until wells are submerged) with electrophoresis buffer (the same buffer used to prepare the agarose)

     Loading the gel: You

     have three dye solutions to run on practice gels.

    One is a diluted solution

     of 5xTAE you normally

     use for PCR reactions.

     Another is a solution containing the same dye

    we normally use, as well

     as a second lighter blue

     dye, which runs more slowly. This solution is in the tube labeled

    BPB/XC. Finally you have chromatrack,

    Student Guide to DNA Fingerprinting by PCRb HELWIG 10

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