Polymerase Chain Reaction (PCR) DNA Amplification
Purpose
To learn the principles and techniques of DNA amplification via polymerase chain reaction (PCR).
Introduction
The polymerase chain reaction (PCR) assay is one of the most important new research tools for biotechnology. Scientists are applying this assay for basic research as well as for applications in pharmaceutical development, forensic investigation, agricultural management, and others. PCR exponentially amplifies specific genetic sequences synthesizing trillions of copies of desired DNA fragments.
DNA polymerase is an enzyme that can synthesize new pieces of DNA using free nucleotide triphosphates (ATP, GTP, CTP, and TTP). The DNA polymerase reads the base sequence of the template DNA strand to be copied and incorporates the corresponding complimentary bases into the growing DNA molecule being synthesized. The DNA polymerase used for PCR can only start synthesizing new DNA from a region of template DNA that is double-stranded at some point. In PCR, the template DNA is first melted by heating to near boiling, creating single-stranded DNA. Then short pieces of DNA called oligonucleotides (typically 15-25 bases long) are added and the temperature lowered enough (to about 50 °C), so that these primers can anneal to (bind to) single-stranded regions with complimentary base sequences. The primers can be synthesized on a machine to have any base sequence and made to specifically match the gene sequences that are to be amplified. Thus, on a very long piece of template DNA, there is only a tiny spot where the DNA is double-stranded, just where the specific primers have bound. And this is the only place primed for binding of the DNA polymerase. The temperature is then raised to the optimal temperature for the polymerase to act (72 °C), and the primer is then extended, generating a new DNA strand, a copy of the gene of interest.
The key to PCR is that this process is then repeated through many cycles, perhaps 40. After one cycle, one target sequence becomes two. After another cycle, the two copies become four copies. This continues through all of the cycles, generating an exponential increase in the number of copies of the desired sequence. The DNA polymerase used for PCR must be able to withstand repeated heating and cooling. Most enzymes would denature (unfold) and become inactivated. The DNA polymerases used for PCR are heat stable because they were isolated from thermophilic bacteria such as Thermus aquaticus found in the hot springs of Yellowstone National Park.
Materials
Gloves DNA Polymerase (red)
Microcentrifuge Tube Rack Buffer Solution (yellow)
8 Empty Micrcentrifuge Tubes Forward Primer (orange)
0.5-10 uL Micropipettor Reverse Primer (orange)
20-200 uL Micropipettor Sterile Water (blue)
Sample DNA (green) Nucleotide Solution (purple)
Procedure
Calculate the Volume of Reagents
Before beginning a PCR reaction the volume of reagents for the reaction master mix must be calculated. You must count how many reactions you need to run (3 in this case) and add 1 additional reaction to your count. By doing this you will be sure to have more than enough master mix to prepare all of the PCR reactions. The total final volume for each reaction will be 50 µl.
DNA polymerase: 1 µL X 4 reactions = 4 µL
Buffer: 5 µL X 4 reactions = 20 µL
Nucleotides: 4 µL X 4 reactions = 16 µL
Forward primer: 2.5 µL X 4 reactions = 10 µL
Reverse primer: 2.5 µL X 4 reactions = 10 µL
Sterile water: 30 µl X 4 reactions = 120 µl
This master mix will then be used to prepare each reaction tube. Each reaction will receive 45 µl of master mix. It will also include:
Genomic DNA sample: 5 µL
Preparing PCR Reactions
1. Label three empty microcentrifuge tubes with the names of the genomic DNA samples.
2. Add 5 µL of genomic DNA sample to each corresponding tube.
3. Label the final empty micrcentrifuge “MM” for Master Mix.
4. Add 120 µL sterile water to the MM tube.
5. Add 20 µL buffer to the MM tube.
6. Add 16 µL nucleotide solution to the MM tube.
7. Add 10 µL of each primer to the MM tube.
8. Add 4 µL DNA polymerase to the MM tube.
9. Gently tap to mix. Do not invert.
10. Add 45 µL Master Mix solution to each of the reaction tubes.
11. Close all of the reaction tubes securely.
12. Give the completed reaction tubes to your teacher. These will be stored frozen until the PCR reactions can be run in a thermocycler. These machines are very expensive, so the reactions will be performed on WSU campus. The products of the reactions will be returned to you for analysis by gel electrophoresis.