The Polymerase Chain Reaction (PCR) is a laboratory technique that allows for the creation of numerous copies of a DNA strand. It takes advantage of the ability of polymerase enzymes to replicate genetic material.
Deoxyribonucleic acid (DNA) plays a fundamental role in various research studies involving living organisms. By examining the DNA code, we can understand the genetic basis of diseases, develop medications, conduct forensic tests, identify microorganisms, and much more.
However, for such research, a significant amount of the DNA fragment under investigation is required. Unfortunately, DNA obtained from cells, tissues, or other biological sources is often insufficient for analysis. Therefore, scientists need to produce additional copies of the DNA.
This is where the “Polymerase Chain Reaction” becomes essential.
What is the Polymerase Chain Reaction?
PCR utilizes the replication ability of polymerase enzymes to generate copies of genetic material in a laboratory setting.
Prior to PCR, DNA copies were produced by isolating a specific DNA fragment and inserting it into the genome of living cells. These living cells would then replicate the inserted DNA along with their own genetic material. This technique was time-consuming and labor-intensive, making it challenging to obtain sufficient DNA copies for further study.
However, thanks to Kary Mullis, who invented PCR in 1983, this is no longer the case. PCR marked the beginning of the “Biotechnology Revolution” and has become a commonly used laboratory technique, even in smaller labs, to regularly generate DNA copies.
PCR can selectively amplify the DNA of interest through a process often referred to as “molecular photocopying”. Once multiple copies of the DNA are synthesized using PCR, the DNA is then amplified.
Polymerase chain reaction (PCR) is an in vitro technique that utilizes DNA polymerase to produce multiple copies of the desired DNA. (Photo Credit: kozhedub_nc/Shutterstock)
What Are The Constituents Of a PCR Reaction?
A PCR reaction consists of several essential components, including template DNA, primers, nucleotides, and heat-stable DNA polymerase. Let’s briefly explore each of these constituents.
PCR can use DNA from various organisms, ranging from simple bacteria to complex animals and plants. However, only a small section of the DNA (known as template DNA) is amplified during the PCR process, not the entire DNA.
For amplification to occur, primers play a crucial role. These are short sequences of nucleotides, approximately 20 base pairs in length. A set of primers, consisting of a forward primer and a reverse primer, is used. These primers attach to the start and end of the DNA strands, indicating the points from which the DNA needs to be amplified.
The nucleotides used in PCR are a mixture of all four nitrogenous bases found in DNA: adenine (A), thymine (T), guanine (G), and cytosine (C).
The final and most important component is DNA polymerase. This enzyme creates new DNA molecules by assembling nucleotides in a way that complements the existing strand. In this manner, it can generate two identical DNA molecules from a single DNA strand.
In addition to the enzyme, cofactors necessary for the activity of DNA polymerase must be included in the reaction mixture. Cofactors are metallic compounds that play a critical role in enzyme function. Magnesium ions serve as cofactors for DNA polymerase.
The DNA polymerase used in PCR is a thermostable enzyme (often referred to as Taq polymerase) isolated from thermophilic organisms capable of surviving high temperatures.
The components of a PCR reaction include template DNA, Taq DNA polymerase, MgCl2 as a buffer, nucleotide bases (dATP, dTTP, dGTP, dCTP), upstream (forward) primer, downstream (reverse) primer, and ultra-pure water. (Photo Credit: Oscar Daniel Luna Ramos/Shutterstock)
How Does a PCR Reaction Work?
After understanding the necessary components for PCR, let’s explore the three main steps involved in a PCR reaction. These steps consist of denaturation, annealing, and extension.
In order for the polymerase to function, single-stranded DNA must be present for the primers to attach to. This can be achieved by heating the DNA sample to a temperature of 94-98°C.
Heating causes the bonds holding the two strands of DNA together to break, resulting in the denaturation of the double-stranded DNA into two separate single-stranded molecules. One of these strands is referred to as the template strand, while the other is known as the complementary strand.
The next step involves the binding (annealing) of the primers to specific sites on the template DNA. The forward primer attaches to the beginning of the template DNA (one of the strands in the double-stranded DNA) at the nucleotide sequence ATG (start codon). The reverse primer attaches to the end of the complementary DNA (the second strand of the double-stranded DNA) at the nucleotide sequences TAG, TAA, or TGA (stop codons). The DNA between the start and stop codons is then amplified.
The success of this step depends on the sequence of the primers and the temperature chosen for annealing, typically ranging from 50-65°C.
The final step in the process is elongation or termination, which occurs at 72°C, the optimal temperature for Taq polymerase. The DNA polymerase recognizes the region of DNA bound by the primer and adds complementary nucleotides to the template DNA strand. This continues until it reaches the second primer.
After a successful termination reaction, there will be two DNA helices instead of the original one used in the beginning. In each helix, one strand will be from the original DNA sample, while the other strand will be synthesized by the DNA polymerase during PCR.
The PCR reaction consists of three steps: denaturation, annealing, and extension or termination. (Photo Credit: Flickr)
How Does DNA Amplification Using PCR Work?
The process of PCR involves three steps: denaturation, annealing, and polymerization. To achieve optimal DNA amplification, a typical PCR reaction may go through 25-35 cycles.
After one cycle, the original single DNA template will produce two DNA molecules. After two cycles, these two molecules will form four DNA molecules, which will then amplify to eight molecules after three cycles. This exponential increase continues, resulting in 2n copies of the initial DNA template after n cycles.
After each cycle, the number of DNA molecules available as templates for the next cycle grows exponentially. This exponential growth is the fundamental principle behind DNA amplification using PCR.
The PCR technique enables the exponential amplification of DNA molecules(Photo Credit: Enzoklop/Wikimedia commons)
Advantages and Disadvantages of PCR
PCR has several advantages as a technique. It is capable of rapidly producing millions to billions of copies of DNA within a few hours. It is also relatively easy to learn and can be performed under basic laboratory conditions.
However, there are also drawbacks to PCR. The technique is highly sensitive, requiring the sample to be free of contaminants. Even small traces of unwanted DNA can be amplified along with the DNA of interest, leading to false results. Additionally, PCR requires sequence information of the DNA to design primers, and there is a risk of non-specific annealing, resulting in the amplification of the wrong DNA fragment. In rare cases, the DNA polymerase may incorporate a wrong base, altering the sequence of the DNA of interest.
To successfully perform PCR, pure genetic material, appropriate primers, and a proper annealing temperature are necessary.
Applications of PCR
PCR has various applications in different fields. It is commonly used for selective DNA isolation from mixed DNA samples, aiding in the diagnosis of infectious diseases, genetic diseases, and certain types of cancers. PCR is also utilized in forensic sciences for criminal identification and paternity testing.
Furthermore, PCR is fundamental to many molecular biology applications, including gene cloning, recombinant DNA technology, and mutagenesis.
Conclusion
The PCR technique, developed by Kary Mullis, has revolutionized biological research. By simply mixing the necessary components in a small tube and using a PCR machine, researchers can obtain millions to billions of copies of the DNA of interest in just a few hours. This rapid and reliable method has greatly improved DNA amplification compared to previous techniques.