Amplification of Genomic DNA
Lesson 7 of 9
Objective: Students will be able to collect a DNA sample from their own cheek cells and use this sample to amplify a gene of interest.
Polymerase Chain Reaction, PCR, is a molecular biology technique that was discovered by Kary Mullis during the early 1980's. The technique uses some clever chemistry applications and creative thermal cycling of the PCR reagents to amplify the gene of interest, the tissue Plasminogen Activator (tPA) gene, located on Chromosome 8. The specific locus targeted for amplification is the Alu element which appears in two forms. An individual can carry a 300bp DNA fragment which carries the Alu element (Alu+) or a 100bp DNA fragment (Alu-) which does not carry this fragment or a combination of either of the forms.
From a single DNA locus, PCR can produce more than one billion copies of the target in less than two hours! This groundbreaking optimization of a process that used to take at least two days to two weeks when I was in graduate school has been so influential that it has given rise to a DNA Technology explosion and earned Kary Mullis the Nobel Prize for Chemistry in 1993.
Students will view the music video by Bio-Rad's - Scientists for Better PCR and sing along! Following the viewing of the music video the instructor will lead the students through a close read and annotation of the "text", which happens to be the lyrics to the PCR song!! Students will also address several focus questions.
For the Student:
Part I - Preparation of Genomic DNA Samples
1. Obtain a 2.0mL screwcap tube containing .5mL of 10% Chelex solution. Using the permanent marker place the initials of the individual being tested or an anonymous code.
2. Use a sterile toothpick or yellow pipette tip to gently scape the inside of both cheeks.
3. Transfer the cells from the toothpick or sterile pipette tip to the Chelex tube by vigorously twirling the toothpick or tip in the Chelex solution to remove the cells collected. (NOTE: Remove as many of the cells off the toothpick or pipette tip as possible. This is an important step.)
4. Close the Chelex tube tightly. Place the tube in a 100C hot water bath or 100C hot block for 10 minutes. This incubation will lyse the cells and help to destroy some of the nucleases, which may degrade the DNA.
5. Centrifuge the tube after removing from the hot water bath or hot block.
6. Label a clean 1.5mL microfuge tube with the initials of the individual being tested or an anonymous code. Using the P-20 pipette and a clean pipette tip, carefully remove 20uL of supernatant and transfer to a clean 1.5mL microfuge tube.
7. If this is the conclusion of the lab block or class session this is a good place to stop. The genomic DNA sample can be kept in the refrigerator at 4C or a freezer at 20C until you are ready to run the PCR reaction.
Part II - Amplification of the tPA Locus
1. Obtain the genomic DNA sample with your number and the PCR tube labeled with your anonymous code number.
2. The PCR tube already contains Master mix I. Master mix I contains the two primers that target the tPA locus, dNTP’s (deoxynucleotide triphosphates: ATP, TTP, CTP and GTP), PCR buffer, molecular grade water (very pure) and Taq polymerase.
3. Using a clean pipette tip, add 5 μL of your genomic DNA to this PCR tube. Carefully add your DNA sample directly into the 10μL of Master mix I. Do this without creating bubbles.
4. Carefully cap the PCR tube. This is a very thin walled tube so avoid crushing it, but make sure that the cap is firmly seated over the opening of the tube.
5. Place your PCR tube into the ice bucket by the thermal cycler.
6. The instructor will add 10μL of Master mix II, containing MgCl2, just before placing your samples into the thermal cycler. Taq polymerase, an enzyme from the bacterium Thermus aquaticus, requires Mg++ ions as cofactors to activate it.
7. Discard your genomic DNA sample.
8. The instructor or designated students will run the PCR reaction at a later time.
9. After the PCR run, 15 μL of your PCR product will be loaded into a 2% agarose gel. The gel will be stained and photo-documented by the instructor.
In their student lab groups students attempt to analyze the results of this lab as seen in this sample or "expected" Lab 8 PCR Result Photo.
In 1993, The Royal Swedish Academy of Sciences awarded the Nobel Prize in Chemistry for contributions to the development of methods within DNA-based chemistry, to Kary Mullis for his invention of the polymerase chain reaction (PCR) method and to Professor Michael Smith, University of British Columbia, Vancouver, Canada, for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies.
The chemical methods that Nobel Laureates Kary B. Mullis and Michael Smith each developed for the study of molecules that serve as the framework of our genetic material rapidly advanced the emerging field of genetic engineering. The two methods have greatly stimulated basic biochemical research and opened the way for applications based on these discoveries to be found in fields such as personalized medicine, biotechnology, and forensic science.
In particular, the applications of Kary Mullis' PCR method are too many to mention in this discussion. However it is important to note that by using a thermocycler, a simple piece of lab equipment able to multiply a given DNA segment millions of times in a few hours, researchers were able to have millions of copies of DNA for biochemical and genetic research. PCR offered new possibilities, particularly in medical diagnostics, was used in the research that led to the discovery of the HIV virus as well as faulty genes in hereditary diseases. Researchers can also produce DNA from animals that became extinct millions of years ago by using the PCR method on fossil material.
Hear first hand in this interview with Kary Mullis’ about the inspirational moment behind the wheel driving in the mountains of Northern California that led to the discovery of PCR!
This lab marks the end of the AMGEN Recombinant DNA Lab Series and the final Lab 8 conclusion questions and Lab 8 PostLab Quiz are the only thing standing between us and future application of all that the Nobel giants have taught us. I like to end the lab series as we began retracing the footstep of the Nobel laureates, such as Kary Mullis, and hoping that we have inspired the next generation of Nobel prize winners due to honoring the work that has earned this supreme honor.