In this blog post, we will examine the innovations brought about by the development of PCR and real-time PCR technology in genetics and molecular biology research, as well as their applications.
In 1993, the Nobel Prize in Chemistry was awarded to Kary Mullis for developing the polymerase chain reaction (PCR). This breakthrough opened the door to amplifying DNA in large quantities even if only a single molecule of DNA with known base sequences was available. PCR requires template DNA, primers, DNA polymerase, and four types of nucleotides. Mold DNA refers to double-stranded DNA extracted from a sample that serves as the basis for DNA amplification in PCR, while the target DNA is the specific region within the mold DNA that is to be amplified. Primers are short single-stranded DNA molecules with nucleotide sequences identical to a portion of the target DNA, with two types of primers binding to the start and end of the target DNA, respectively. DNA polymerase replicates DNA by sequentially binding nucleotides corresponding to each base sequence of single-stranded DNA to form double-stranded DNA.
This process has brought about a major innovation in molecular biology and biotechnology research. The development of PCR has become an essential tool for various biological research and medical diagnostics. For example, it is widely used in various research fields such as gene cloning, gene expression analysis, mutation analysis, and gene mapping. It also plays an important role in forensic science, such as paternity testing and DNA evidence analysis at crime scenes.
The PCR process begins by applying heat to separate the double-stranded DNA into two single strands. Then, primers bind to each single-stranded DNA, and DNA polymerase replicates them, creating two double-stranded DNA molecules. This DNA replication process, which occurs over a set period of time, forms one cycle, and the amount of target DNA doubles with each cycle. The PCR is terminated once the cycle has been performed sufficiently to prevent further amplification of DNA.
Traditional PCR detects the amplification of target DNA by binding fluorescent substances to the final PCR product and observing color changes. This led to the groundbreaking development of real-time PCR, which can also determine the amount of target DNA in a sample. Real-time PCR performs PCR in the same way as traditional PCR but incorporates a color reaction at each cycle, allowing the accumulation of color to monitor the amplification of target DNA in real time.
For this purpose, real-time PCR requires an additional fluorescent substance during the PCR process, such as “double-stranded DNA-specific dyes” or “fluorescent-labeled probes.” Double-stranded DNA-specific dyes are fluorescent substances that bind to double-stranded DNA and emit light, enabling the detection of target DNA amplification by binding to newly formed double-stranded target DNA. However, double-stranded DNA-specific dyes can bind to all double-stranded DNA, so if two primers bind to each other to form a double-stranded dimer, unintended color development occurs due to binding to this dimer.
Fluorescent-labeled probes are single-stranded DNA fragments consisting of a fluorescent substance and a quencher that suppresses the fluorescence of the fluorescent substance. They are designed to specifically bind to regions of target DNA where primers do not bind. During PCR, when double-stranded DNA is converted into single strands, the fluorescent-labeled probes bind to the target DNA in the same manner as primers. During the subsequent process of DNA polymerase-mediated double-strand formation, the probe is cleaved from the target DNA and degraded. When the probe is degraded, the fluorescent material and quencher are separated, causing the fluorescent material to emit light, thereby indicating that the target DNA has been amplified. Fluorescent-labeled probes have the advantage of specifically binding to the target DNA but are relatively expensive.
In real-time PCR, the intensity of fluorescence is proportional to the amount of amplified double-stranded target DNA, and the number of cycles required to reach a certain level of fluorescence varies depending on the initial amount of target DNA. The change in fluorescence intensity over the course of the cycles is displayed as a continuous line, and the number of cycles required to reach the fluorescence 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 in the unknown sample can be calculated.
PCR is widely used for various applications, including genetic replication, genetic disease diagnosis, paternity testing, and diagnosis of cancer and infectious diseases, using DNA obtained from samples. In particular, real-time PCR enables accurate and rapid diagnosis of viral infections at an early stage. Additionally, various application studies utilizing PCR technology are currently underway. For example, PCR is used to investigate biodiversity in ecosystems through environmental DNA analysis and to develop new therapies targeting specific genes in gene therapy research.
In this way, the development of PCR has brought significant advancements in the field of life sciences, and its potential applications remain vast and untapped. PCR technology continues to evolve, with new variations and applications being researched on an ongoing basis. As a result, the importance and usefulness of PCR are expected to grow even further across various fields.
