The polymerase chain reaction represents a procedure from molecular biology that duplicates sections from genetic material (deoxyribonucleic acid, DNA). Millions of identical copies are produced from minute amounts of DNA. In this way, quantities are available that are sufficient for various investigations.
What is the polymerase chain reaction?
The polymerase chain reaction represents a method from molecular biology that duplicates sections from genetic material (deoxyribonucleic acid, DNA). The term polymerase chain reaction (PCR) describes the in vitro (Latin: in the glass) reaction with the help of an enzyme, the polymerase (DNA polymerase), which leads to the duplication of certain gene sequences. The product of the reaction is also the starting material for a new cycle of this reaction. The number of molecules doubles and at the same time serves as a template for a new cycle. This is referred to as exponential multiplication. It takes place in the laboratory at a great speed of a few minutes, similar to a chain reaction. This laboratory process mimics the duplication of genetic information (DNA) that occurs under natural conditions during replication. The American chemist K. B. Mullis is considered the discoverer of this process. In 1983, he introduced this DNA synthesis process and ten years later was awarded the Nobel Prize in Chemistry.
Function, effect, and goals
In living organisms, the DNA in chromosomes has a length that cannot be amplified using PCR. Instead, it is applied to amplify a defined section. This can be genes, a specific part of a gene, or regions that are not transcribed into proteins, i.e., are non-coding. These sections usually comprise no more than three thousand base pairs, compared to approximately twice three billion base pairs per set of chromosomes in humans. The polymerase chain reaction requires a single- or double-stranded DNA chain whose structure must be at least partially known. In addition to the enzyme, the polymerase, two primers are used. These are building blocks of the DNA that act as the start and end point. They are characterized by a sequence that exactly matches the region to be amplified. In the laboratory, the polymerase chain reaction is carried out in a programmable heating block. The necessary components such as polymerase, primer, the building blocks to build the new strand (deoxyribonucleoside triphosphates) and magnesium ions are added together in a buffer solution. The temperature-time program for the reaction starts with denaturation at a temperature above 94°C. In this process, the double-stranded DNA is cleaved and is present in single-stranded form. In the next step, at about 70°C, the primer is bound to the gene sequence and forms the starting point for the enzyme reaction. From here, the polymerase synthesizes the complementary strand. A new cycle then begins, again consisting of the three steps of denaturation, primer binding and synthesis of the DNA strand. The polymerase chain reaction is used in forensic medicine, clinical diagnostics and clinical research. In forensic science, DNA is extracted from skin, saliva, hair, semen or blood from crime scenes and, after amplification, compared with known samples and used to identify specific individuals. Using this genetic fingerprint, paternity can also be clarified in a modified approach. In the elucidation of diseases, the polymerase chain reaction is used to verify the genes involved. Some bacterial diseases can be classified by recognizing specific sequences. Viral diseases can be characterized when the viral DNA or RNA is transformed and amplified. In blood screening, it is possible to detect hepatitis or HIV-mediated diseases at a very early stage. In tumor diagnostics, it is used to identify tumor cells. This makes it possible to classify the tumor, assess the course of the disease, the success of the therapy and the prognosis. In research, the polymerase chain reaction is used to identify genes associated with various diseases.For gene cloning, which is not the same as cloning an organism, the gene is amplified before being transferred to other organisms in a vector (Latin: traveler, carrier). These can act as models to better study disease or to produce proteins that can be used as drugs.
Risks and hazards
The polymerase chain reaction has huge potential in detecting minute amounts of DNA. In order to take advantage of the multitude of possibilities and to guard against momentous errors, certain prerequisites and various sources of error must be taken into account. Only sections of the genetic material whose sequence is at least partially known can be amplified. Completely unknown sequences cannot be amplified by this method. The products of an amplification can be visualized afterwards. If the expected signal is not visible, although the sought sequence was present, a false negative result is present. Most often, this is the result of non-optimized or poorly optimized reaction conditions. These must be determined as a function of the target sequence. For this purpose, different temperature and time profiles, primer sequences and amounts as well as concentrations of other substances in the reaction mixture are tested. False positive results show up as signals that cannot be assigned to the desired product. Major problems are caused by contaminations with DNA originating from the investigator or from a source other than the one to be analyzed. DNA of bacterial origin also influences the result of a polymerase chain reaction. By wearing gloves and taking great care, such errors can be prevented and reliable conclusions about the amplified products can be formulated.
