PCR has had a transformative effect on biology because it can amplify extremely small quantities of DNA into quantities that can be analyzed. Previously, the only way to increase the amount of DNA was by utilizing bacterzia to create more copies. This process was time-consuming and involved transferring a DNA fragment into a plasmid or another vector, introducing the combined DNA into a bacterial cell, growing large quantities of bacteria, and then purifying the DNA fragment from the bacteria once again. With PCR, specific DNA sequences can be rapidly generated in large amounts without the need to transfer the DNA into bacteria to produce the copies. This makes it easier to purify the DNA fragments.
Unlike living organisms, PCR is limited in its ability to copy only short DNA fragments, typically up to 10 kilobase pairs (kb) in length. Some techniques can copy fragments as long as 40 kb, which is still much shorter than the chromosomal DNA of a eukaryotic cell. For instance, a human cell has about three billion base pairs, far exceeding the maximum length of DNA that PCR can copy.
PCR has become an essential tool in the research laboratory for a variety of purposes, such as genetic cloning, sequencing, and measuring gene expression. Additionally, it is utilized in medicine to accurately identify microbes, including individual pathogens or groups of diverse organisms, like those found in the gut microbiota.
In medical and biological research laboratories, PCR is a frequently utilized technique for various purposes including the identification of hereditary diseases, establishing genetic fingerprints, diagnosing infectious ailments, cloning genes, performing paternity testing, and DNA computing.
How Does the PCR Process Work?
PCR necessitates the presence of several fundamental components, which include:
- A DNA template or cDNA that contains the segment of the DNA fragment to be amplified.
- Two primers that identify the starting and ending points of the section to be amplified
- Taq polymerase, which duplicates the segment to be amplified.
- Nucleotides, which serve as the building blocks for the new DNA strands synthesized by the DNA polymerase.
- A buffer solution, which offers an appropriate chemical environment for the DNA polymerase to function.
PCR reactions are conducted in a thermal cycler, which is a device capable of precisely heating and cooling the reaction tubes within it to the appropriate temperature for each stage of the reaction. To prevent the reaction mixture from evaporating, a heated lid is positioned on top of the reaction tubes, or a layer of oil is added to the surface of the reaction mixture.
Before delving into the process of PCR, it is important to have a thorough understanding of its fundamental principles. PCR relies on four nucleotide bases, namely Adenine, Thymine, Cytosine, and Guanine, to amplify high-quality DNA fragments, even from limited sources. Typically, a single PCR cycle involves 30-35 repetitions and takes about two hours to generate a usable amount of DNA fragments. By incorporating slight variations, such as multiplex PCR, RT-PCR, Nested PCR, inverse PCR, colony PCR, asymmetric PCR, helicase PCR, ligation-mediated PCR, etc., PCR can be customized to produce better results. The PCR reaction involves 20-40 cycles, each with three different temperatures.
PCR comprises of three crucial steps per cycle, which include denaturation, primer annealing, and final extension.
Denaturation
The process of denaturation in PCR involves heating the DNA sample to around 95°C to separate the double-stranded DNA into single-stranded DNA by breaking down the hydrogen bonds between the two complementary bases. This denaturation phase typically takes 15-30 seconds in a standard PCR reaction.
Primer Annealing
The second step is called annealing, where the temperature is lowered to around 60°C. This allows the primers to attach to the complementary sequences in the DNA sample, and the temperature is held for about 15-60 seconds, depending on the length of the primers. Longer primers are more specific in binding to the exact target sequence. The annealing temperature is low enough to enable the primer bases to form hydrogen bonds with the target DNA, and it is determined based on the sequence of the target DNA and primers.
Final Extension
The third step is elongation or extension, where the temperature is raised to the ideal temperature for the thermostable DNA polymerase being used, which is typically about 72°C. At this temperature, the DNA polymerase uses nucleotides to extend or elongate the primer, creating a new complementary DNA strand. The duration of the extension phase depends on the length of the target DNA, with longer targets requiring more time for the DNA polymerase to create a complete copy.
The PCR process can be adapted depending on its intended application in both biology and medicine. For instance, researchers have developed group-specific PCR strategies such as Touchdown PCR and LAMP assays that allow for more targeted detection of specific sequences regardless of minor differences between them. These adaptations have proven useful for applications such as Zymo microbial community analysis and microbial disease diagnosis.
Applications of PCR in Biology and Medicine
One of the primary applications of PCR in medicine is rapid diagnosis. Researchers have been able to use PCR to rapidly identify viruses like HIV, herpes simplex virus (HSV-2) as well as fungal species like Candida albicans, helping doctors confirm diagnoses faster than traditional testing methods. In fact, due to its flexibility and accuracy PCR has become an indispensable tool for identifying pathogens relevant in clinical settings.
The high sensitivity of PCR also helps it stand out from other laboratory techniques when diagnosing rare diseases where the genetic mutation associated with the disease may only occur in a few cells.
Besides detecting pathogen-causing agents, PCR can be used to assess gene expression levels in a sample by quantifying levels of mRNA present. This provides useful information about cellular health and can aid medical professionals to diagnose cancers or severe illnesses.
In the context of biological research, PCR still plays a fundamental role in modern-day labs around the world because it provides insight into how organisms evolved and how organisms interact with each other. It’s also useful for analyzing previously uncharacterized genes or pathways as well as for screening mutants out of populations. Thanks to its numerous practical applications, countless scientists have been using PCR since its introduction three decades ago — making it one of the most powerful molecular tools researchers today have at their disposal.
Detecting DNA Sequences in Research and Diagnosis
PCR has been a valuable tool for use in detecting DNA sequences in both research and clinical diagnosis. Historically, restriction enzymes have enabled researchers to detect and analyze various genetic abnormalities, but PCR can provide much more precise results with lower amounts of sample material. PCR does come with some drawbacks that must be weighed when considering its use for a given application. Since the method relies on obtaining the specific sequence of DNA to amplify, any changes within the target sequence may render the results useless or inaccurate. Additionally, due to its specificity, findings should always be further confirmed with other assays or sequencing methods such as Sanger sequencing. Perhaps most important though is considering the cost compared with alternative methods—sometimes other processes may be more suitable depending on the resources available.
The implications of PCR in detecting DNA sequences are far-reaching and are proving to be pivotal in advancing research and diagnostics. These advances provide vast opportunities for greater knowledge and precision in understanding genetic composition and identifying conditions that might have otherwise gone unnoticed.
References
- (PDF) Polymerase Chain Reaction (PCR): A Short Review (researchgate.net) The Discovery of PCR: ProCuRement of Divine Power – PMC (nih.gov)
- Polymerase Chain Reaction. Molecular Biology, 168–198 | 10.1016/B978-0-12-813288-3.00006-9
- Clark, D. P., Pazdernik, N. J., & McGehee, M. R. (2019). Polymerase Chain Reaction. Molecular Biology, 168–198. doi:10.1016/b978-0-12-813288-3.00006-9
- different_types_of_pcr_techniques_and_its_applications.pdf (ksu.edu.sa)