Technical Description

Abstract

     The polymerase chain reaction (PCR) is a tool used by scientists to create multiple specific strands of deoxyribose nucleic acid (DNA). The tool was first created by Kary B. Mullis, Ph.D 1983 and since then has gone through multiple advancements such as an improved DNA Taq polymerase and a better thermal cycler for a faster and more efficient process. To conduct PCR, the target DNA sequence is needed, along with DNA Taq polymerase, deoxynucleotide triphosphates (dNTPS), DNA primers, a thermal cycler, and a buffer solution containing cations. The process of PCR is composed of three steps, denaturing, annealing, and elongation. Denaturing causes the DNA strands to break apart in the thermal cycler. During annealing, DNA primers are added to the complementary strands. Afterward, elongation allows DNA polymerase to build a complementary strand from the primers. This process can be repeated multiple cycles, with each cycle producing 2ⁿ strands of DNA, where n is the number of cycles. PCR is used in a wide variety of applications including testing for diseases in the medical field and criminal evidence in forensic science. PCR is a simple process that has had a huge impact on the scientific world and will continue to do so.

Introduction

     The polymerase chain reaction (PCR) is a tool used by biologists to amplify a certain sequence of deoxyribose nucleic acid (DNA). This technical description serves to detail the history and the process of PCR in the scientific field. PCR is important for scientists and biologists because it allows them to obtain copies of DNA without having to rely on a single or small amount of DNA. Copied DNA can be used in a multitude of ways to serve another function. PCR has allowed various fields like forensic science and genealogy to make advancements, as well as understand our genome through experiments. 

 History

     The polymerase chain reaction was first conceived in 1983 by Kary B. Mullis, Ph.D. Mullis was a scientist working in Cetus Corporation. For 2 years, Mullis and his team researched and refined the process of PCR to make the idea a reality. In 1985, the team of scientists presented at the American Society for Human Genetics meeting and later that year published their results in Science, a journal of the American Association for the Advancement of Science (“History” 1). However, the first version of the PCR process had a lot of problems. Not only was the process slow, but it required every cycle in the process to be reset manually. It included a DNA polymerase, an enzyme that synthesizes DNA from nucleoside triphosphates, from a bacterium called E. coli. At that time, PCR required constant heating and cooling to break the hydrogen bonds holding together the two strands of DNA. However, E. coli was unable to perform efficiently at high temperatures and constantly needed to be replaced manually every cycle. The team of scientists at Cetus Corporation even created a thermal cycler called “Mr. Cycle” to rapidly heat and cool the DNA. It required a new set of DNA polymerase from E. coli every cycle (“History” [Smithsonian]1). But in the following years, several advancements allowed PCR to be faster and more autonomous. The first advancement was Taq DNA polymerase. Like DNA polymerase from E. coli, Taq DNA polymerase is an enzyme found in the bacterium Thermus aquaticus that allows the enzyme to synthesize DNA from nucleoside triphosphates. The benefit is that Taq DNA polymerase functions better at higher temperatures than E. coli DNA polymerase since it thrives in high temperature environments. It removes the need to manually reset the polymerase every cycle. The second advancement was a thermal cycler produced by Perkin Elmer. Their thermal cycler allowed the regulation of temperatures in the reactions during PCR and minimized labor requirements (“History” 1). These advancements allowed PCR to evolve into the tool biologists know and use today in labs. 

Requirements

     PCR requires various components for it to work. The first thing is the target DNA. The target DNA is the specific sequence of DNA that you want to replicate. DNA takes the shape of a double helix and is composed of two complementary strands that are held together by hydrogen bonds and run anti-parallel to one another. Each strand is composed of a series of nucleotides. A nucleotide is a molecule that is composed of a phosphate group, sugar molecule, and one of the four nitrogenous bases (adenine, guanine, cytosine, or thymine). The second thing is the DNA polymerase. DNA polymerase is an enzyme that elongates a pre-existing sequence of DNA strand by adding nucleotides to it. The most common DNA polymerase that is used in PCR is Taq DNA polymerase because of its’ ability to continuously function at high temperatures of 90 degrees Celsius. Third, deoxynucleotide triphosphates (dNTPs) are needed as the building blocks of the DNA copies. The dNTPs contain one of the four nitrogenous bases and the “concentration must be in excess and may need to be increased for amplification of long fragments or highly abundant targets” (“Polymerase” 1). Fourth, two sets of DNA primers are needed, the forward primer and the reverse primer. Primers are short sequences of DNA and are only a couple base pairs long. They bind to the complementary DNA strands and allow DNA polymerase to add dNTPs to them. Fifth, a thermal cycler is needed to monitor and control the temperature of the solution during PCR. Sixth, PCR requires a buffer solution. A buffer solution is a liquid that contains cations like K+ or Mg2+ to help in the synthesis of the new DNA strand. There should be at least 1 biologist with knowledge on PCR monitoring and conducting the process. Furthermore, the process must be done in a controlled environment, like a lab, where environmental variables, like temperature, can be regulated. Each quantity of components will depend on the scale of production and the proportion to other components.

Process

     The polymerase chain reaction is made up of three steps, denaturing, annealing, and elongation. Every time the three steps are completed, one cycle of PCR has been done. PCR starts with the first step called denaturing. During this step, biologists take the template DNA and place it in a buffer solution. The buffer solution contains cations that provide the template DNA with a stable chemical environment by keeping the pH between 8.0 and 9.5. Afterward, biologists place the buffer solution with the template DNA inside a thermal cycler. There, the temperature of the thermal cycler is raised to about 90 degrees Celsius to break the hydrogen bonds between the two strands in DNA. This causes the DNA strands to split from one another. This step takes about 30 seconds (“What” 1). At this point, the first step is finished, and the DNA is denatured. 

     In the second step, biologists anneal the separate strands of the template DNA. They add the forward and the reverse primers in the buffer solution of the DNA strands. Because the temperature is still hot, the two strands of DNA have not reformed their hydrogen bonds with one another. As a result, the two strands of the template DNA are open to bond with the primers. Once the primers are added, the biologist then cools the temperature of the thermal cycler down to about 50 to 56 degrees Celsius. This allows the hydrogen bonds between a strand of the template DNA and a primer to form through polymerization. The second step takes about 30 seconds. Once the primers are bonded to the template DNA, the second step is completed, and the annealing of the primers and the DNA strands is finished.

     In the final step, elongation, biologists use Taq DNA polymerase to elongate the primers. The temperature is increased to 72 degrees Celsius inside the thermal cycler and biologists add Taq DNA polymerase and dNTPs into the solution. The Taq DNA polymerase recognizes the primers attached to each of the DNA strands and begins to build a new DNA strand by adding dNTPs to the primers from the 5’ to 3’ direction. “72⁰ C is the optimum temperature for the Taq polymerase to build the complementary strand” (“What” 1). The temperature is low enough to not break the hydrogen bonds and high enough to allow the Taq DNA polymerase to function in a similar high-temperature environment. “The duration of this step depends on the length of DNA sequence being amplified but usually takes around one minute to copy 1,000 DNA bases (1Kb)” (“What” 1). Taq DNA polymerase stops constructing the DNA strand when it reaches a sequence of DNA that signals it to stop or if it runs out of DNA to make a complementary strand. At the end of the elongation, the temperature is brought back down to 50 degrees Celsius, and the DNA has two strands again. Thus, one cycle of PCR is completed after the elongation.

Results

     At the end of PCR, the result is double the copies of the DNA. The relationship between the number of cycles and the number of DNA copies produced can be determined by using the function f(n) = 2^n, where n is the number of cycles. The f(n) equals the number of DNA copies after n cycles. Because it is an exponential function, the change in the number of DNA copies every cycle becomes greater every time a cycle is completed. This allows replication of small quantities into large quantities of DNA in a small amount of time. 

Applications

     Because of polymerase chain reaction’s ability to replicate a small amount of DNA exponentially over a small period, it is used in a wide variety of sciences and in conjunction with other processes. In medical applications, PCR can be used to amplify a small amount of DNA from the patient to diagnose a disease based on the primers that bind to the template DNA. In mutagenesis, biologists can alter and study mutations in an organism by changing the primers and manipulating the dNTPs. In real-life applications, PCR can be used in forensic science to capture criminals or exonerate convicted felons. PCR can amplify the polymorphic sites of an individual’s DNA, and since each polymorphic site is unique to the individual, it can be used to profile individuals for crimes. Other real-life applications include genealogy, the study of ancestry. The “same polymorphic sites used by criminalists to explore the involvement of a suspect in a criminal act are also used by testing laboratories to identify the father of a child whose parentage is in dispute” (“Forensic” 2). PCR allows paternity testing and can help identify an individual’s biological mother or father. 

Conclusion

     The polymerase chain reaction is an important tool for scientists and biologists to allow them to amplify DNA sequences for the real world and laboratory experiments. By following the process, biologists will be able to make many copies of the template DNA from a few original strands. PCR has come a long way from being inefficient and slow. Through advancements like thermal cycler and the discovery of Taq DNA polymerase, PCR is not only more efficient, but it is faster and cheaper to conduct. Under the guidance of a scientist or biologist, PCR can be used to solve criminal and paternity cases, map ancestry, and study the genome of organisms. 

Works Cited

“Forensics and Paternity.” Calculations for Molecular Biology and Biotechnology, by Frank Harold Stephenson, Elsevier/AP, Academic Press Is an Imprint of Elsevier, 2016, www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/applications-of-pcr.

History of PCR, Roche, diagnostics.roche.com/global/en/article-listing/history-of-pcr.html. 

“The History of PCR.” Smithsonian Institution Archives, Smithsonian Institution,

siarchives.si.edu/research/videohistory_catalog9577.html. 

“Polymerase Chain Reaction: PCR Process & Guide.” Sigma, www.sigmaaldrich.com/technical-

documents/articles/biology/polymerase-chain-reaction.html. 

“What Is PCR (Polymerase Chain Reaction)?” Facts, The Public Engagement Team at the

Wellcome Genome Campus, 25 Jan. 2016, www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction.