Cary Mullis’ polymerase chain reaction (PCR) technology, which won the Nobel Prize in Chemistry in 1993, provided a way to amplify a single DNA molecule into large quantities, revolutionizing gene cloning, disease diagnosis, forensics, and more. PCR uses template DNA, primers, DNA polymerase, and nucleotides to amplify DNA, and real-time PCR allows for the amplification of target DNA to be viewed in real time. This technology is particularly important for rapidly diagnosing viral infections.
In 1993, the Nobel Prize in Chemistry was awarded to Kary Mullis for the development of the polymerase chain reaction (PCR). His work paved the way for amplifying large quantities of a single molecule by knowing its sequence. 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, and two primers bind to the beginning and end of the target DNA. DNA polymerase replicates DNA by joining the nucleotides corresponding to each sequence of bases in the single-stranded DNA in sequence to create double-stranded DNA.
The PCR process begins by applying heat to separate the double-stranded DNA into two single strands. A primer then 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, where the amount of target DNA in the sample is also known. 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, which is called a “double-stranded DNA-specific dye” or “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 allows for early, accurate, and rapid diagnosis of viral infections. This can play an important role in pandemic situations and can make a significant contribution to public health. Recent advances in PCR technology have also led to its use in a variety of other fields, including environmental monitoring, agriculture, and biotechnology research. For example, PCR is used to monitor changes in microbial communities in environmental samples or to genetically improve crops.
PCR technology has also become an important tool in forensic science. It can be used to amplify microscopic DNA samples collected from crime scenes to identify criminals or to search for missing persons, among other forensic applications. As you can see, PCR technology has become an essential tool in many areas of science, and its importance is expected to grow in the future.
