Lab 1: Who s Your Daddy? (AKA DNA Purification and PCR)

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1 Lab 1: Who s Your Daddy? (AKA DNA Purification and PCR) Goals of the lab: 1. To understand how DNA s chemical properties can be exploited for purification 2. To learn practical applications of DNA purification techniques 3. To perform DNA amplification by PCR 4. To be introduced to proper pipetmen use Introduction: Deoxyribonucleic Acid (DNA) is the genetic material of all cellular organisms on the planet and contains the instructions that specify their biological development. While the sequence of DNA between two different humans is over 99% identical, that small difference can have dramatic effects. As you have likely seen in the media, these differences can be used to determine family lineages and aid in criminal investigations. In the research laboratory, the manipulation of DNA is used to study biological processes. Two techniques that have helped advance biological knowledge are the cloning of DNA and its propagation, and the amplification of specific DNA sequences. Both of these techniques will be described more fully below. This week you will learn two fundamental techniques to manipulate DNA. You will purify DNA and use PCR to determine the specific genetic makeup (genotype) for a single gene in a human DNA sample. Your purified plasmid DNA will be used in PCR reactions as a control to differentiate the two different forms (alleles) of a gene in the human DNA sample. HIV infection The retrovirus HIV infects different types of cells in the body. The ability of HIV to enter cells is determined largely by its binding to two proteins on the surface of the cell it is trying to infect. These proteins are called a receptor and a coreceptor, which you will learn more about in lecture. For HIV to enter a cell it binds to a receptor on the surface of the cell called CD4 and a co-receptor. There are many different strains of HIV and the cell types that are infected by HIV are determined mainly by the co-receptor that HIV binds along with CD4 to enter cells. Two of the major co-receptors for different HIV strains are CXCR4 and CCR5. Interestingly 10% of the Caucasian population harbors a mutant form of CCR5 where the gene is 32 nucleotides shorter (CCR5Δ32). In approximately 1% of the Caucasian population, two copies of the mutant form of CCR5 are present. 26

2 The presence of two copies of the mutant CCR5 reduces their rate of infection from some strains of HIV. NOTE: Presence of this mutation in a person s genome DOES NOT EQUATE immunity from HIV-1. It only suggests that the rate of HIV-1 infection in this person is lower against some HIV-1 strains, not all. Cloning From science fiction and the popular media, the most common assumption about cloning is that it is the production of a genetically identical organism. For instance, in 1997 a clone of a sheep was generated from the cell of another adult sheep. More commonly in molecular biology, cloning refers to the copying of a gene or part of a gene. In this lab, you are purifying human DNA that has been cloned into a smaller piece of circular DNA so it can be replicated in bacteria. In this lab, the two bacterial cultures you are provided with contain clones of CCR5. One tube contains a clone of wildtype CCR5 and the other CCR5Δ32, both kindly provided by Dr. Philip Murphy and Dr. S. Venatesan of the NIH. DNA Purification (mini-prep) The principle of the mini-prep DNA purification is to isolate small, circular DNA from bacteria. Most scientists today use kits (as you will today), and the exact nature of how these kits enable you to purify DNA is often proprietary. However several fundamental aspects are identical in all DNA purification techniques. 1. Lyse the bacteria. The DNA must be released from the inside of the cell. Lysis can be achieved in many different ways, however the two most common are through the use of heat (boiling) and chemically (change in ph, and detergents). 2. Separation of cellular components away from DNA. Another chemical is added that will allow the DNA to stay in solution, however the other cellular components will precipitate (form a solid) out of the solution. A centrifuge can then be used to separate the solid portions from the solution. 3. Purification. The DNA is often present in a large volume with many salts and other small molecules. One can take advantage of the chemical properties of DNA (the negative charge) to separate it from other remaining components. PCR The polymerase chain reaction has fundamentally changed molecular biology. With its development, DNA can be manipulated and amplified from a single copy to millions of copies in a matter of hours. The powerful amplification that can occur with PCR has benefits and drawbacks. A benefit is that one can amplify very limited quantities of DNA rapidly. A drawback is that very small amounts of 27

3 DNA contaminants are also capable of being amplified. If you pay attention to court cases involving DNA evidence, attorneys will often question the handling techniques of biological samples to ascertain if any contaminating DNA could have been inadvertently introduced. PCR can be a complicated procedure requiring the precise addition of multiple reagents. However advances have greatly simplified the procedure and allowed for a substantial amount of automation. One of the advances has been the prepackaging of many PCR components into different forms. In this lab you will be using PCR beads where the salts, nucleotides (dntps) and thermostable enzyme have been packaged into a dried bead. The only components the experimenter must add are primers, a DNA template to amplify, and water. Pipetmen For quantitative experiments, it is essential that the experimenter is able to measure weights, lengths, and volumes reproducibly and accurately. One of the great inventions for molecular biology was the invention of the digital micropipettor. When used properly, these devices allow for the rapid and reproducible transfer of specific volumes of liquids from 1 ml to less than 0.5 µl. In this lab, you will be using digital micropipettors to add specific volumes of various reagents for your mini-prep procedure and PCR. Please watch the pipettor video prior to class. You will use pipettors of several different sizes in the lab. They may be marked , , and These numbers indicate the effective range in microliters (µl) that these pipetmen can transfer. Never attempt to transfer a volume outside of the effective range of a pipettor. Reagents: Mini-prep PCR Gel Electrophoresis 1 ml bacterial culture kit components Buffer P1 Buffer P2 Buffer N3 Buffer PE Buffer EB Sterile water Water/Primer mix Gel PCR products DNA loading buffer 100 bp DNA ladder (Molecular weight marker) 28

4 Protocol: Week 1 Procedure for Qiagen mini-prep Each group will receive two 1 ml bacterial cultures to perform 2 mini-prep DNA purifications. 1. Each lab pair will receive one tube containing CCR5 and CCR5Δ32 bacteria. 2. Resuspend the bacterial pellet completely by pipetting the solution up and down with 250 µl of P1 buffer 3. Add 250 µl of P2 buffer. Cap your tube and invert 4-6 times and incubate for 5 min at room temperature. Your solution should become viscous and turn blue. 4. Add 350 µl of buffer N3 and mix by inverting 4-6 times. A fluffy white precipitate should form, and the blue color should completely disappear. If your solution is still blue, continue inverting until the solution becomes clear. 5. Spin your tube for 10 min in a microfuge at maximum speed 6. While your tube is spinning, label two Qiagen spin columns for your two mini-preps. 7. Using a P1000 micropipettor, remove the supernatant and add it to your labeled Qiagen spin column. Make sure not to transfer any of the white, flocculent precipitate. 8. Spin the tube for 1 min. 9. Discard the liquid in the collection tube and add 750 µl of buffer PE to the column and spin again for 1 min. 10. Discard the liquid and spin your column for 1 min (no liquid is added in this step). 11. Place your spin-column into a new, labeled 1.5 ml microfuge tube and add 50 µl of buffer EB to your column and incubate for 1 min at room temperature. Make sure that the liquid contacts the bottom of the column. a. Label your tubes with the date, your initials, and the plasmid name (either CCR5 or CCR5Δ32) EC-EJH CCR5Δ Spin your column for 1 min. After the spin, you should have approximately 50 µl of mini-prep purified DNA in your microfuge tube. 13. Prepare two new tubes and dilute a small amount of your plasmid DNA 100-fold with sterile water. 29

5 Procedure for PCR Isolation of human DNA sample: In this portion of the lab, you will obtain a DNA sample from your cheek cells that will be analyzed for the genotype of CCR5. The sample you will analyze will not be your own, but a random sample from the class. If you do not wish to provide a DNA sample, another sample will be provided for you. 1. Swab the inside of your cheek with a sterile swab for a few seconds. 2. Scrape the swab in an unlabeled microfuge tube with 500 µl of Phosphate Buffered Saline (PBS), and cap the tube. 3. Give your tube to your TF and he/she will centrifuge your sample along with those of your classmates for 1 minute at 13,000 RPM. 4. Your TF will return a randomized sample to you to perform the rest of the experiment. 5. Label your tube with your initials 6. Remove the PBS and resuspend the pellet with 50 µl of PBS. 7. Transfer the resuspended cells and PBS into a small (0.5 ml) tube. 8. Heat your sample for 5 minutes at 100 C on a heatblock (PCR machine). 9. Spin down the tube for 1 min to pellet cellular debris. The supernatant contains the DNA. PCR: PCR will be performed during the class, and the results will be analyzed next week. 1. Each student will obtain 2 PCR tubes from their TF and label them on the top and side with their initials and the number 1 or To each tube, add 20 µl of primer/water (red solution) mix a. Once added, your primers will be present at a final concentration of 1 µm each. 3. To tube #1 add 5 µl of genomic DNA sample, to tube #2 add 5 µl of diluted mini-prep DNA. a. Make sure to note in your notebook whether your mini-prep DNA is from CCR5 or CCR5Δ32 4. Place your tubes on ice and give them to your TF to perform the PCR. 5. Your TF will place your tubes in the PCR machine and run the reaction when all of the class s tubes are ready. 30

6 The PCR will be performed with the following parameters: Initial DNA denaturation: 95 C 5 min 35 cycles of the following temperatures and times: 95 C 30 s 68 C 30 s 72 C 30 s Final polymerization step to complete the synthesis of any partially synthesized DNAs: 72 C 7 min Storage: 4 C 24 hours Week 2 Analysis of PCR: You will run a gel to separate the DNA produced in the PCR reaction by size. Smaller pieces of DNA will migrate more quickly (move further) on the gel than larger pieces of DNA. Your TF will show you how to load the gel. In this particular lab you will be using a 2% Agarose E-gel. There are 12 wells on each E-gel, and 3 groups will be running their sample per gel. One of the three groups will not run one of their positive control (plasmid) samples and instead will load the molecular weight marker. 1. In your notebook, record what sample you will load in which lane 2. Load 10 µl of 100 bp DNA ladder in lane 1 3. Load 20 µl of each reaction into individual lanes on the gel 4. Run the gel at 70V for approximately 30 minutes, observing the migration of dye. 5. Once the dye has migrated 2/3 of the length of the gel, turn off the power supply and ask your TF to see if your gel has run long enough. 6. Take a picture of the gel and print two copies for your personal notebooks. 31

7 Questions: 1. Attach a labeled copy of your results (picture of your gel), and describe them. a. What did you do in the experiment? b. What are the sizes of the DNA products? c. Which band corresponds to which DNA? d. Are there bands for your genomic DNA samples? If not, why? e. How many bands are present in your genomic DNA samples? f. Which alleles do they represent? g. Why did you run PCR reactions from your plasmid DNA samples? h. Are there single or double bands in your plasmid DNA samples? What do both situations mean? i. Are there any bands in PCR tube #5? If there are, what does that mean? 2. Three different temperatures were used during the PCR reaction. What is occurring in the tube at each of these temperatures? 3. In this experiment, the PCR primers were 100% complementary to the DNA sequence in the genomic samples. If the primers used were instead 80% complementary, would any of the temperatures used in the PCR reaction have to be changed? Which one and why? 4. Given the following genotypes, what proteins will be expressed in T cells, and can they be infected by HIV? Genotype T-cell expression HIV infectivity CCR5/CCR5 CCR5/CCR5Δ32 CCR5Δ32/ CCR5Δ32 5. Genomic DNA was isolated mostly from epithelial cells lining your mouth. Would the PCR reaction have worked more efficiently or robustly if T-cells were instead used as the source of DNA? Explain. 6. Scientists can also look at RNA expression inside cells by PCR. Would the PCR results analyzing RNA versus DNA be identical for all cell types? Explain. 7. Does the PCR experiment done today indicate whether or not CCR5 protein is expressed in the cheek tissue sample? 8. In the Qiagen mini-prep you performed, DNA binds to an anion (negatively charged) binding column at specific ph and salt conditions. To remove DNA from the column, a low salt concentration wash is used. a. How can the DNA be bound to an anion column? 32

8 b. How does a low-salt concentration wash cause DNA to be released from the column? 9. Given the reagents you were provided with in this laboratory, could you determine the presence or absence of HIV-1 in an unknown sample? Explain. 10. Design a PCR based method for detecting HIV-1. 33

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