In 1993, Nobel Prize winner in Chemistry Mullins developed the polymerase chain reaction (PCR), which revolutionized DNA amplification. PCR is widely used in a variety of fields, including DNA cloning, genetic research, and disease diagnosis. Real-time PCR technology, in particular, allows the process of DNA amplification to be monitored in real time. Advances in PCR technology have had a profound impact on genetics and molecular biology research, and the possibilities for future applications are endless.
The 1993 Nobel Prize in Chemistry was awarded to Mullis for his development of the polymerase chain reaction (PCR). It paved the way for the amplification of large amounts of DNA from a single molecule of sequenced DNA. PCR requires template DNA, primers, DNA polymerase, and four nucleotides. The template DNA is the double-stranded DNA that is extracted from the sample and is the basis for DNA amplification in PCR. The region of the template DNA that you want to amplify is called the target DNA. Primers are short, single-stranded pieces of DNA with the same sequence as a portion of the target DNA. Two primers bind to the beginning and end of the target DNA, respectively. DNA polymerase replicates DNA by joining nucleotides corresponding to each sequence of bases in single-stranded DNA in sequence to create double-stranded DNA.
This process has revolutionized molecular biology and biotechnology research. The development of PCR has made it an essential tool for a wide range of biological research and medical diagnostics. For example, it is widely used in a variety of research fields, including gene cloning, gene expression analysis, mutation analysis, and genetic mapping. It also plays an important role in forensic science, where it is used to determine paternity and analyze DNA evidence at crime scenes.
The PCR process begins with the application of heat to separate the double-stranded DNA into two single strands. A primer binds to each single strand of DNA, which is then replicated by DNA polymerase to create two double strands of DNA. This process of DNA replication, which takes place over a period of time, is called a cycle, and the amount of target DNA doubles with each cycle. The PCR is terminated after enough cycles have been performed to ensure that the amount of DNA is no longer amplified. Traditional PCR binds a fluorophore to the end product of the PCR and uses color to determine whether the target DNA has been amplified.
PCR has led to a breakthrough development called real-time PCR, which can also determine the amount of target DNA in a sample. Real-time PCR performs the same PCR as traditional PCR, but allows the color reaction to occur in each cycle, so that the accumulated coloration can be used to determine the amplification of target DNA in real time. To achieve this, real-time PCR requires an additional chromogenic agent to be added to the PCR process, either a “double-stranded DNA-specific dye” or a “fluorescently labeled probe. Double-stranded DNA-specific dyes are fluorophores that bind to double-stranded DNA and fluoresce when they bind to the newly generated double-stranded target DNA, thus indicating the amplification of the target DNA. However, double-stranded DNA-specific dyes can bind to any double-stranded DNA, so if two primers bind together to form a double-stranded dimer, this can cause unintended coloration.
A fluorescently labeled probe is a single-stranded DNA fragment with a fluorophore and a quencher that inhibits the fluorophore, designed to specifically bind to sites on the target DNA where primers do not bind. When double-stranded DNA becomes single-stranded during PCR, the fluorescently labeled probe binds to the target DNA just as the primers do. During the subsequent formation of double-stranded DNA by DNA polymerase, the probe loses its binding to the target DNA and degrades. Only when the probe is degraded and the fluorophore and the quencher are separated does the fluorophore emit light, indicating that the target DNA has been amplified. Fluorescently labeled 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 chromogenicity over the course of the cycle is shown as a continuous line, and the number of cycles required to reach the chromogenicity at which the target DNA is judged to be detected is called the Ct value. By comparing the Ct value of an unknown sample, where the concentration of target DNA is unknown, to the Ct value of a standard sample, where the concentration of target DNA is known, the concentration of target DNA in the unknown sample can be calculated.
PCR is widely used to clone genes, diagnose genetic diseases, determine paternity, and diagnose cancer and infectious diseases using DNA obtained from samples. In particular, real-time PCR can be used to accurately and quickly diagnose viral infections at an early stage. In addition, various applications of PCR technology have been developed in recent years. For example, PCR is used to analyze environmental DNA to investigate the biodiversity of ecosystems, or to develop new therapies that target specific genes in gene therapy research.
As you can see, the development of PCR has been a major advance in the life sciences, and the possibilities are endless. PCR technology is constantly evolving, and new variations and applications are constantly being researched. This is expected to increase the importance and usefulness of PCR in various fields.
