In this blog post, we will introduce how PCR technology is used in various fields, such as forensic evidence analysis and environmental microbial monitoring.
In 1993, the Nobel Prize in Chemistry was awarded to Kary Mullis for his development of the polymerase chain reaction (PCR). His achievement paved the way for the amplification of large quantities of DNA from a single molecule. PCR requires template DNA, primers, DNA polymerase, and four types of nucleotides. Mold DNA is double-stranded DNA extracted from a sample that serves as the basis for DNA amplification in PCR, and the part of the mold DNA to be amplified is called target DNA. Primers are short single-stranded DNA molecules with the same base sequence as part of the target DNA, and two types of primers bind to the beginning and end of the target DNA, respectively. DNA polymerase replicates DNA by binding nucleotides corresponding to each base sequence of single-stranded DNA in order to produce double-stranded DNA.
The PCR process begins by applying heat to separate the double-stranded DNA into two single strands. Then, when primers bind to each single strand of DNA, they are replicated by DNA polymerase to form two double strands of DNA. This DNA replication process, which takes place over a set period of time, constitutes one cycle, and the amount of target DNA doubles with each cycle. The PCR is terminated after a sufficient number of cycles have been performed to prevent further amplification of the DNA. In traditional PCR, a fluorescent substance is added to the final product of PCR to check whether the target DNA has been amplified by color development.
PCR led to a revolutionary development called real-time PCR, which can also determine the amount of target DNA in a sample. Real-time PCR is performed in the same way as traditional PCR, but a color reaction occurs at each cycle, and the accumulation of color can be used to confirm the amplification of the target DNA in real time. For this purpose, real-time PCR requires an additional color-forming substance, such as a “double-stranded DNA-specific dye” or a “fluorescent probe.” A double-stranded DNA-specific dye is a fluorescent substance that binds to double-stranded DNA and produces a color reaction. It binds to newly formed double-stranded target DNA and produces a color reaction, indicating the amplification of the target DNA. However, since double-stranded DNA-specific dyes can bind to all double-stranded DNA, if two primers bind to each other and form a double-stranded dimer, unintended coloration will occur due to binding to this dimer.
A fluorescent probe is a single strand of DNA to which a fluorescent substance and a quenching substance that suppresses this fluorescent substance are attached, and is designed to bind specifically to regions of the target DNA where primers do not bind. During the PCR process, when double-stranded DNA becomes single-stranded, the fluorescent probe binds to the target DNA in the same way as the primer. Subsequently, during the process of double-stranded DNA formation by DNA polymerase, the probe is separated from the target DNA and degraded. When the probe is degraded and the fluorescent substance and quenching substance are separated, the fluorescent substance finally emits light, indicating that the target DNA has been amplified. Fluorescent probes have the advantage of binding specifically to the target DNA, but they are relatively expensive.
In real-time PCR, the color intensity is proportional to the amount of amplified double-stranded target DNA, and the number of cycles required to reach a certain color intensity varies depending on the initial amount of target DNA. Changes in color intensity as the cycle progresses are displayed as a continuous line, and the number of cycles required to reach the color intensity at which the target DNA is detected is called the Ct value. By comparing the Ct value of an unknown sample whose target DNA concentration is unknown with the Ct value of a standard sample whose target DNA concentration is known, the concentration of target DNA contained in the unknown sample can be calculated.
PCR is widely used for gene replication, genetic disease diagnosis, parentage testing, and cancer and infectious disease diagnosis using DNA obtained from samples. In particular, real-time PCR can be used to accurately and quickly diagnose viral infections at an early stage. This can play an important role in pandemic situations and contribute greatly to public health. Recently, advances in PCR technology have led to its increasing use in various fields such as environmental monitoring, agriculture, and biotechnology research. For example, PCR technology is used to monitor changes in microbial communities in environmental samples and for research on genetic improvement of crops.
In addition, PCR technology has become an important tool in forensic science. It can be used in various forensic applications, such as amplifying minute DNA samples collected at crime scenes to identify perpetrators and searching for missing persons. As such, PCR technology has become an essential tool in various fields of science, and its importance is expected to grow even further in the future.
