Polymerase chain reaction
The polymerase chain reaction, abbreviated as PCR by AbbreviationFinder is a molecular biological method for amplification of DNA segments. The method is now one of the standard procedures in molecular biology and was developed in 1985 by KB Mullis (* 1944, † 2019) . KB Mullis received the Nobel Prize in Chemistry for this in 1993.
This is how PCR works
The process of a PCR runs in cycles that comprise three steps:
- Denaturation: During denaturation, the DNA, which consists of two strands, is broken down into its individual strands by increasing the temperature to more than 90 ° C. This makes it accessible for reproduction.
- Binding of a primer: A primer is a short DNA sequence that can bind to its exact counterpart on the single strand of DNA, thereby defining the starting point of the PCR. In addition, the primer also limits the area of the DNA that is replicated. For this step, the temperature is lowered to around 55 to 65 ° C.
- DNA production: The DNA is extended starting from the primer. This process is carried out by a heat-stable enzyme called polymerase. This causes certain DNA sequences to be duplicated. The primer is not removed, but remains part of the new strand.
By repeating these three steps several times, a million-fold enrichment of a certain DNA sequence is achieved. In the laboratory, a run with the appropriate equipment takes a few minutes.
Areas of application
PCR is used whenever there is insufficient DNA material available by another route. Even the smallest traces of DNA are sufficient to generate a sufficient amount of a DNA sequence for various analyzes and purposes via the PCR.
The PCR is used in many areas, for example in research, in medicine, for genome analysis for hereditary diseases, for relationship tests or paternity tests or in forensic medicine to solve crimes (analysis of the genetic fingerprint).
Polymerase chain reaction
Polymerase chain reaction, abbreviation PCR [ of English p Polymerase c hain r eAction], by the American biochemist KB Mullis developed molecular genetic method for reproduction of DNA sequences.
PCR was developed as a molecular genetic method for the replication (amplification) of DNA sequences.
Through a step-by-step process, certain DNA sequences can be enriched millions of times, either from an entire genome or from a pool of different DNA sequences. The underlying principle is a repetition of three successive steps: denaturation, primer annealing and elongation.
When the DNA is denatured, the typical double-stranded form of the DNA is separated by heating to 95 ° C, which means that the melting temperature of the DNA is significantly exceeded. The DNA is then present in its single strands and is accessible for replication.
The primer annealing
The specificity of the DNA amplification is ensured by adding so-called primers. Primers are DNA sequences that are complementary to the sequences in front of and behind the DNA to be replicated and have a length of 10 to 30 bases (also called nucleotides). The primers thus form the start and end point of the DNA amplification and ensure that only a certain DNA sequence is enriched during the PCR. In order to bind the primers to the single strands of DNA, the temperature is lowered to 50-60 ° C. The exact annealing temperature depends on the length and sequence of the primers. It is just below the melting point of the primer sequences.
The elongation / DNA synthesis
In the next step, the DNA sequence, based on the primers, is produced complementary to the existing DNA single-stranded sequence. Nucleotides that are added to the reaction in excess are added to the primers. This happens according to the base sequence of the DNA single strand that serves as a template. This process is catalyzed by the enzyme polymerase, which adds the individual nucleotides to the growing DNA sequence. The temperature is increased to 72 ° C, which corresponds to the optimal temperature of the Taq polymerase used (polymerase from the bacterium Thermus aquaticus). At the end of this synthesis, one double-stranded DNA has become two identical double-strands.
This cycle (denaturation – primer annealing – DNA synthesis) is now repeated 30 to 40 times, each time doubling the DNA. This results in a millionfold enrichment of the desired DNA sequence. Thus, even the smallest traces of DNA are sufficient to generate a sufficient amount of a DNA sequence for various analyzes and purposes via the PCR.
The PCR takes place in a so-called thermal cycler, an apparatus in which the rapid heating and cooling takes place automatically during the individual cycles. The success of the amplification can then be determined by examining the size of the DNA sequence by means of gel electrophoresis.
Areas of application
The PCR method is now of great importance as a molecular biological method for a wide variety of areas. In genetic engineering, PCR is a standard method for amplifying certain DNA sequences, which can then be transferred to target organisms in a suitable form.
PCR is also used in many areas of medicine, such as for diagnosing genetic defects (also in prenatal diagnostics) and for the rapid identification of pathogens. PCR tests, inter alia for HIV, the hepatitis B virus, chlamydia, SARS-CoV-2 and numerous other pathogens are now routine methods in medical diagnostics. In addition, statements about the course of a virus infection and also about the course and success of the therapy are possible with the help of the PCR analysis.
However, PCR is also used in other areas. In forensics, it is used to determine the genetic fingerprint and can lead to the investigation of criminal offenses. In addition, the PCR is used in paternity tests and in evolutionary biology or archeology to determine lineages.
The PCR method was developed by KB Mullis in 1985. In 1993 was awarded the Nobel Prize in Chemistry for the invention.
As with many other technical achievements, a creative trick was required, without which the current status of PCR would be inconceivable. In order to make the DNA double strand single-stranded, the DNA solution must be heated to over 90 ° C. The original version of the polymerase chain reaction provided that the DNA polymerase had to be added for each synthesis cycle. This was necessary because enzymes usually lose their activity after exposure to heat – a process that is as expensive as it is impractical given the high price of purified enzymes.
The decisive advance was the use of a DNA polymerase from a heat-stable bacterium called Thermus aquaticus. Heat-stable ( thermophilic or hyperthermophilic) bacteria colonize unusual habitats, e.g. B. volcanic springs in the deep sea. In order to survive here, they are adapted to the high temperatures of their environment by structural features – they have enzymes that can withstand heating to over 90 ° C without loss of activity. The DNA polymerase from Thermus aquaticus, the so-called Taq polymerase, fulfills this requirement. Since this no longer has to be pipetted in again before each new cycle, the entire process of DNA amplification can be simplified and automated. Theoretically, a single DNA fragment can be copied billions of times within a few hours. In analogy to the processes involved in nuclear fission, the term “chain reaction” was also chosen here.
Due to its great economic importance, the polymerase chain reaction quickly became a patent precedent in biotechnology. Both Taq polymerase and PCR technology are protected by patents, and it is believed that a major pharmaceutical company paid $ 300 million for these patents alone.