The machine in Figure 2 (LEFT) is made by Stratagene and is their RoboCycler®. The Stratagene RoboCycler® on the other hand
1. OVERVIEW OF PCR METHODOLOGY
The polymerase chain reaction (PCR) was developed in order to amplify DNA. It mimics DNA replication, a naturally occurring process in living cells. Kary Mullis, a biochemist, conceived the method in 1983 and later developed the method along with his associate, Fred Faloona at the Cetus Corporation in Emeryville, CA (1, Encarta). PCR is a technique that "widens the boundaries of inquiry in fields as diverse as medical research and diagnostics, forensics, genetics, microbiology, evolutionary biology, and industrial technology" (99,Marini). Minor changes have been made with regard to the procedure in order to increase the efficiency of it.
One of the steps taken to improve the technique was the introduction of Thermus aquaticus as the bacterium from which the enzyme, Taq polymerase is obtained. This particular enzyme is heat stable at 95o C, therefore it can withstand the denaturation step used in the PCR method. The Taq polymerase, however, does not have a proofreading mechanism; therefore there are far more mistakes made than when the previously used bacterium, E.coli was used.
The versatility of PCR is amazing; it will amplify anything granted it can be flanked with a pair of primers and powerful enough to start the process with a one-molecule sample. The number of DNA segments increase exponentially, essentially doubling in number after each cycle. Thirty PCR cycles are capable of producing 1 billion DNA copies in less than 3 hours.
There are three phases involved in the polymerase chain reaction. All of the phases take place in the same test tube containing the double-stranded DNA of interest; a large enough supply of A,T,C, and G bases which are the building blocks of DNA; a pair of primers and a polymerase such as Taq polymerase. The first phase is called denaturation and involves the heat denaturation of double-stranded target DNA. The test tube is heated to nearly boiling for a few seconds. This step makes the DNA separate into two single strands. At this point the primers can bind to the single strand where the sequence is complementary to that of the DNA. Phase two involves the annealing of specific primers to target DNA. The temperature of the test tube contents is lowered to 55oC. The DNA is now mainly single-stranded with a few double-stranded areas in which primers have aligned themselves. The third phase involves primer extension by the action of DNA polymerase. At this point the temperature is raised again, this time the temp. is raised to 72oC for about 1 minute in order to allow the Taq polymerase to perform its function. The polymerase moves along the single-stranded DNA and creates a second strand of new DNA to match the targeted sequence. These steps are repeated about 20-30 times before new "ingredients" are added to the test tube to begin the process again. Completing the cycle 30 times will fill the test tube with the fragment of DNA that only accounted for 1:3,000,000 in the original sample.
PCR eliminates loads of tedious work that scientists previously had to perform. One no longer has to search for genetic markers for diseases. With PCR, a single strand of DNA can make billions of the same needed DNA sequence. The process is very simple to perform. After the needed materials are all added to the same test tube, they simply need to be placed in a device known as a thermal cycler that performs the temperature changes necessary for PCR to take place. There are a number of companies that market thermal cyclers. The machine that is shown in Figure 1 (LEFT) is marketed by Snark Technologies and is the Snark 96. Most often, the biggest downfall with PCR is the price. The prices of some thermal cyclers seem to have come down over the years. The Snark 96 is one such example, with simplified functions and lower costs.
The machine in Figure 2 (LEFT) is made by Stratagene and is their RoboCycler®. The Stratagene RoboCycler® on the other hand
Though the cost has oftentimes been considered a disadvantage, the uses usually outweigh the cost. Now, with the more basic thermal cyclers, even smaller laboratories can afford to use them. As previously mentioned, PCR is useful for many different applications, such as AIDS testing, DNA fingerprinting (to help ID criminals), genetic diagnostics, gene therapy, etc.
Marini, R.A. 1992. Polymerase Chain Reaction. Popular Science. V240:n5. p99(5).
Encarta Encyclopedia. Polymerase Chain Reaction. Ó 1993-1998 Microsoft Corporation.
Madigan, M.T., J.M. Martinko and J. Parker. 1997. Brock Biology of Microorganisms. Pp.374-77.
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