In this blog post, we will look at the innovations and applications that the development of PCR and real-time PCR technology has brought to genetics and molecular biology research.
The 1993 Nobel Prize in Chemistry was awarded to Mullis, who developed the polymerase chain reaction (PCR). This is because it has opened the way to amplify a large amount of DNA if there is even one molecule of DNA whose base sequence is known. PCR requires template DNA, primers, DNA polymerase, and four nucleotides. Template DNA refers to double-stranded DNA extracted from a sample and used as the basis for DNA amplification in PCR, and the region to be amplified from template DNA is called target DNA. Primers are short single-stranded DNA molecules that have the same base sequence as a portion of the target DNA, and two primers bind to the beginning 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 produce double-stranded DNA.
This process has brought about major innovations 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 the field of forensics and is used for paternity testing and analysis of DNA evidence at crime scenes.
The PCR process begins by first heating to separate the double-stranded DNA into two single strands. Then, when a primer binds to each single-stranded DNA, it is replicated by a DNA polymerase to form two double-stranded DNAs. This process of DNA replication, which takes place over a certain period of time, forms a cycle, and the amount of target DNA doubles with each cycle. And the PCR is terminated after the DNA has been sufficiently amplified to no longer be amplified. Traditional PCR confirms whether the target DNA has been amplified by binding a fluorescent substance to the final product of PCR and detecting the color development.
PCR led to the development of a revolutionary real-time PCR that can also determine the amount of target DNA in a sample. Real-time PCR performs PCR in the same way as traditional PCR, but it allows the color reaction to occur every cycle, so the amplification of target DNA can be checked in real time through the accumulated color. To do this, real-time PCR requires an additional color-developing substance in the PCR process, which is achieved using “double-stranded DNA-specific dyes” or “fluorescent label probes.” Double-stranded DNA-specific dyes are fluorescent substances that bind to double-stranded DNA and develop color, binding to newly generated double-stranded target DNA and developing color, which allows the amplification of target DNA to be detected. However, since the double-stranded DNA-specific dye can bind to all double-stranded DNAs, it binds to two primers that have combined to form a double-stranded dimer, causing an unintended color reaction.
A fluorescent-labeled probe is a single-stranded DNA fragment with a fluorescent material and a quenching material that suppresses this fluorescent material, and is designed to specifically bind to a site on the target DNA where the primer does not bind. During the PCR process, when double-stranded DNA becomes single-stranded, the fluorescent label probe binds to the target DNA, just like the primer. Then, during the process of forming double-stranded DNA by the DNA polymerase, the probe loses its binding to the target DNA and is degraded. When the probe is decomposed and the separation of the fluorescent and quenching substances occurs, the fluorescent substance is finally colored, indicating that the target DNA has been amplified. Fluorescent labeling probes have the advantage of binding specifically to 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 level of color intensity depends on the initial amount of target DNA. The change in color intensity over the course of the cycle is displayed as a continuous line, and the number of cycles required to reach the color intensity at which the target DNA is considered to have been detected is called the Ct value. The concentration of target DNA in an unknown sample can be calculated by comparing the Ct value of the unknown sample, for which the concentration of target DNA is unknown, with the Ct value of a standard sample, for which the concentration of target DNA is known.
PCR is widely used to replicate genes, diagnose genetic diseases, identify parentage, and diagnose cancer and infectious diseases using DNA obtained from samples. In particular, real-time PCR can be used to accurately and quickly diagnose the presence of a virus at an early stage. In addition, various applications of PCR technology are currently being studied. For example, PCR is used to investigate the biodiversity of ecosystems through environmental DNA analysis, and to develop new treatments targeting specific genes in gene therapy research.
The development of PCR has brought about significant progress in the life sciences field, and its potential for use is endless. PCR technology continues to evolve, and new variations and applications are being continuously researched. As a result, the importance and usefulness of PCR in various fields is expected to increase.
