Polymerase Chain Reaction (PCR)

Sam Krug English 202C 3/26/14

Background Information Polymerase Chain Reaction (PCR) is a laboratory technique that is used to help amplify specific sections of DNA. These strands of DNA are then able to be analyzed and put into new cells to see how their behavior is affected. This technique is mostly used when a cell is being designed that will use genes from multiple sources. DNA is able to replicate on its own, but it takes several hours to occur. PCR is able to optimize the temperature and process that is needed for replication to happen so that instead of taking several hours in order for one copy to be made, thousands can be made in the same amount of time. There are specific materials that are needed in order to run a PCR and each reaction follows the same three steps; denaturation, annealing, and extension. Necessary Materials In order to have a successful experiment, it is important that all of the necessary parts of a PCR reaction are present. All of the components will be mixed in a small tube called a PCR reaction tube. First and foremost, a copy of the DNA is needed. This will serve as the template so that many copies can be formed from this original strand. PCR also requires a forward and backward primer. Primers indicate where DNA should start to replicate. There is an extensive process to design these primers, but there are computer programs that are capable of designing them. By having both forward and reverse primers present in the reaction tube, it allows the reaction to hone in on a specific segment of DNA; instead of copying a strand of hundreds of thousands of base pairs, it will only result in several hundred to a couple thousand being replicated. The amount of time needed to reproduce a similar strand of DNA will reduce drastically as it becomes shorter. Also needed is a piece of lab equipment called a Thermocycler. This machine is capable of being programmed to change temperatures after certain periods of time. Without this machine, PCR would be nearly impossible. The Biology behind the Process PCR consists of three major steps: denaturing, annealing, and extension. Combined, these three elements are called a cycle. PCR is usually carried out using 25 cycles to ensure the maximum amount of DNA will be produced. This figure shows the temperatures and time that each step takes during a cycle. They must be in this order for the reaction to work properly. There are specific characteristics of each step that are necessary for to enhance DNA replication and each step helps to lead to the next.

Denaturing Denaturing the DNA is the first step in PCR. The word denature means to modify a structure and in this case, the structure is DNA. To initiate the cycle, DNA is modified to lose its shape and come out of its double helix structure. This results in having two individual strands of complementary DNA. This is achieved by raising the temperature of the solution to 95 degrees Celsius so that the forces holding the DNA in its helix shape are disrupted. This only occurs for a short period of time because if DNA is denatured for too long, it may never regain its shape. The separate strands give the primers in the solution an opportunity to bind to their corresponding spot. Annealing The primers used are designed to complement specific sections of the DNA strands. In the annealing phase of a PCR cycle, the primers are able to attach to the DNA because the temperature of the solution is lowered to 50 degrees Celsius. The length of the annealing phase of the PCR cycle is usually around one minute because this will allow the primers enough time to bind to the appropriate part of the DNA and will reduce the chance of primers incorrectly matching themselves with another part of the DNA strand. Once the primers are attached, the cycle enters the extension phase. Extension During the final phase of PCR, the complementary strand of DNA is assembled so that there will be double the number of DNA strands than there were to begin with. The primers alert DNA where to start replicating so after the primers have bound to the DNA, replication can begin. Since DNA synthesis occurs faster at higher temperatures, the Thermocycler heats the solution to 70 degrees Celsius. This is a high enough temperature that it optimizes DNA synthesis, but it is not hot enough for the strands of DNA to denature. This step is slightly longer than the others to ensure that the entire DNA segment is synthesized. T his step usually lasts around two minutes, but can be adjusted to be longer if a longer segment of DNA is trying to be replicated. Once this phase is over, the solution can be heated up to 95

degrees Celsius again to start another cycle, or the finished products can be removed from the Thermocycler. Final Products At the end of the cycles, the solution will have many copies of the same DNA segment. This has many different applications in the lab. One of the most common uses is for new genes to be inserted into cells so that they will behave a certain way. It is also common for PCR to be used in police work in order to replicate DNA evidence that is found at a crime scene. PCR is applicable to many different fields of work and has helped to further knowledge of main different studies since its invention. Bibliography for Pictures Brown, Terry A. Gene Cloning and DNA Analysis. 6th ed. Hoboken: Wiley-Blackwell, 2010. Print.