Alu - PCR (Polymerase Chain Reaction):
Background: Humans are unique in the sense that we are bipedal and think like no other creatures. We share a common language, communicate complex ideas, and flock together to do great things. Compared to other animals, like fish and birds and even primates, we could not be more different. But beneath our physical features and our associated intelligence, we humans share one fascinating building block with every other species on this planet: deoxyribonucleic acid (DNA). Over the years, the human genome has been concluded to hold approximately 3 million base pairs distributed on 23 chromosomes; all of our body's cells, except erythrocytes, sperm, and eggs, contain these 46 chromosomes. Yet only 15% of this DNA is used directly to code for proteins required for supporting cellular metabolism, growth, and reproduction; and these protein-encoding regions, called exons, are scattered few and far between each other. 90 to 97% of the human genome are made up of introns -- hundreds of thousands of base pairs intervening between exons that, interestingly enough, have not been proven to code for anything. Because they have no defined role, introns have been referred to as "Junk DNA" by scientists. Closer examination revealed that there were repeating elements in these non-coding regions; one element identified was Alu. It's approximately 300 base pairs in length, and mostly carries the base sequence AGCT, which is the recognition site for Alu I restriction endonuclease, a type of enzyme that cuts DNA at a specific site. Alu repeats occur around every 4,000 base pairs along a human DNA molecule. Scientists believe that the first Alu appeared in the genome of higher primates about 60 million years ago; every 100 years since then, a new Alu repeat has inserted itself in an additional location in the human genome. Because it's been proven that Alu repeats are inherited in a stable manner and come intact in the DNA your mother and father contribute to your genome, the Alu repeats could prove a significant tool in the molecular clock and in trying to track down our ancestors and how humans evolved.
What is PCR?
PCR, or the Polymerase Chain Reaction, is a method used by scientists to rapidly copy, in vitro, specific segments of DNA. By mimicking some of DNA replication strategies employed by living cells, PCR has the capacity to make millions of copies of a particular DNA region. It takes a small segment of DNA and amplifies it so that scientists can closely study it.
Stage 1: Denaturing Two strands of template DNA molecules are separated, or "denatured," by exposure to a high temperature (94-96* C). Once the bases of the template DNA are exposed, they are free to interact with the primers.
Stage 2: Annealing The reaction is brought down to a temperature around 37-65*C, which allows stable hydrogen bonds to form complementary bases of the primers and template. The primers require only seconds to locate and anneal to their complementary sites.
Stage 3: Extension The reaction temperature is raised to 65-72*C. The DNA polymerase starts adding nucleutides to the ends of the annealed primers. These three phases repeat over and over again, doubling the number of DNA molecules with each cycle.
PCR, or the Polymerase Chain Reaction, is a method used by scientists to rapidly copy, in vitro, specific segments of DNA. By mimicking some of DNA replication strategies employed by living cells, PCR has the capacity to make millions of copies of a particular DNA region. It takes a small segment of DNA and amplifies it so that scientists can closely study it.
Stage 1: Denaturing Two strands of template DNA molecules are separated, or "denatured," by exposure to a high temperature (94-96* C). Once the bases of the template DNA are exposed, they are free to interact with the primers.
Stage 2: Annealing The reaction is brought down to a temperature around 37-65*C, which allows stable hydrogen bonds to form complementary bases of the primers and template. The primers require only seconds to locate and anneal to their complementary sites.
Stage 3: Extension The reaction temperature is raised to 65-72*C. The DNA polymerase starts adding nucleutides to the ends of the annealed primers. These three phases repeat over and over again, doubling the number of DNA molecules with each cycle.
Purpose: The purpose of this experiment was to:
1. Be able to list and explain the importance of each component of PCR
2. Compare PCR to cellular DNA replication
3. Associate the temperature changes with the cycling steps of PCR
1. Be able to list and explain the importance of each component of PCR
2. Compare PCR to cellular DNA replication
3. Associate the temperature changes with the cycling steps of PCR
Materials:
- 0.9% saline solution - Micropipettes, tips - waste container - microcentrifuge - microcentrifuge tubes |
- PCR tubes - agarose - 1XTAE - loading dye - gel chambers and molds |
- chelex - racks - primer mix - master mix - water and control DNA |
Procedure:
1. Swirl 10 ml of saline solution in your math for 30 seconds. Expel saline into a cup and swirl to mix cells.
2. Transfer 1000 microliters of the saline/cell suspension into your labeled microfuge tube. Spin in a microcentrifuge to pellet the cells. Pour off the supernatent, allowing 100 microliters to cover the cell pallet. Rack the sample.
3. Withdraw 50 microliters of your cell suspension and add it to a tube containing Chelex.
4. Apply Chelex tube to a heat block for 10 minutes.
5. Remove Chelex tube from heat block. Use a P-200 to withdraw 50 microliters of supernatent from the Chelex tube and transfer to a fresh tube.
6. Obtain a tiny PCR tube and keep on ice.
7. Pipette 20 microliters of Master Mix into the PCR tube. Then add 20 microliters of Primer Mix.
8. Add 10 microliters of your extracted DNa into the PCR tube.
9. Place reaction into a thermal cycler.
10. Retrieve PCR tube and spin in a microcentrifuge. Then, add 5 microliters of loading dye.
11. Create and pour gels. Add 1XTAE solution.
12. Load 15 to 20 microliters of the DNA/loading dye mixture into a well in your gel.
13. Load 5 to 10 microliters of a 100 base-pair ladder (molecular weight marker) into the one well in each gel for later comparison.
14. Run gels.
1. Swirl 10 ml of saline solution in your math for 30 seconds. Expel saline into a cup and swirl to mix cells.
2. Transfer 1000 microliters of the saline/cell suspension into your labeled microfuge tube. Spin in a microcentrifuge to pellet the cells. Pour off the supernatent, allowing 100 microliters to cover the cell pallet. Rack the sample.
3. Withdraw 50 microliters of your cell suspension and add it to a tube containing Chelex.
4. Apply Chelex tube to a heat block for 10 minutes.
5. Remove Chelex tube from heat block. Use a P-200 to withdraw 50 microliters of supernatent from the Chelex tube and transfer to a fresh tube.
6. Obtain a tiny PCR tube and keep on ice.
7. Pipette 20 microliters of Master Mix into the PCR tube. Then add 20 microliters of Primer Mix.
8. Add 10 microliters of your extracted DNa into the PCR tube.
9. Place reaction into a thermal cycler.
10. Retrieve PCR tube and spin in a microcentrifuge. Then, add 5 microliters of loading dye.
11. Create and pour gels. Add 1XTAE solution.
12. Load 15 to 20 microliters of the DNA/loading dye mixture into a well in your gel.
13. Load 5 to 10 microliters of a 100 base-pair ladder (molecular weight marker) into the one well in each gel for later comparison.
14. Run gels.