PCR stands for Polymerase Chain Reaction, which was invented in the 1980s. The principle of PCR depends on DNA polymerase. In this process, replication of specific DNA in an in-vitro environment is performed.
A particular fragment of DNA i.e. the targeted DNA will create tens of billions of copies from a DNA extract. Through PCR, it is possible to selectively replicate the sequence of interest in a DNA (in large numbers) if it is present in the DNA extract.
Below, we discuss the principle of PCR to get you familiarized with the process.
Multiple copies of a DNA fragment can be obtained by in-vitro replication of DNA in a skirted PCR plate. Matrix DNA can be obtained by RT-PCR as genomic DNA or complementary DNA from a messenger RNA extract, as well as mitochondrial DNA. From a DNA sample, you can obtain large amounts of specific DNA. But how do you amplify the sample? By double-stranded DNA replication. The process involves a denaturation phase, hybridization phase and elongation phase.
A reaction mixture comprising DNA extract, Taq polymerase, the primers and four deoxyribonucleoside triphosphates is used to perform the polymerase chain reaction. The mixture in the tubes is subjected to several temperature cycles in the heating block of a thermal cycle. This apparatus allows a succession of different temperature cycles in each step, and programming of the duration is also made possible. A period of tens of seconds for each cycle is required. The process of PCR is divided into three stages, which are described as follows.
In this step, the DNA is separated into two strands by raising the temperature. The denaturation temperature for DNA is 94℃. DNA matrix is denatured at this temperature, as the hydrogen bonds can not survive at temperatures that go above 80℃. Hence, the double-stranded DNA is converted into single-stranded DNA.
Hybridization is the second step in a PCR. For this step, the temperatures are maintained between 40 to 70℃. This temperature is known as the primer hybridization temperature. Hydrogen bonds can reform as we lower down the temperature, and hence, the complementary strands can hybridize. The short single-stranded sequences which flank DNA to be amplified are called primers. These are easily hybridized as compared to the long strand matrix DNA. If you need more selective hybridization, you should opt for higher temperatures, as they provide more specific results.
Elongation is carried out at a temperature of 72°C. In this step, a complementary strand is synthesized. At this temperature, primed single-stranded DNA and Taq polymerase bind with each other and work as a catalyst in the replication using deoxyribonucleoside triphosphate from the reaction mixture.
Thus, the regions of template DNA downstream of primers are synthesized selectively. The fragments synthesized in the previous cycle are the matrix in the next cycle. After completion of a few cycles, the predominant species correlates with DNA sequence between the region of primers hybridization. Synthesis of an analyzable amount of DNA is achieved after 20-40 cycles. The final cycle of elongation is recommended to be at a temperature of 72℃ in case the sequence of interest is large.
What Are Primers?
It is required to have at least one pair of oligonucleotides for the selective amplification of nucleotide sequences obtained from a DNA extract. These oligonucleotides are synthesized chemically and serve as primers for replication. For the amplification of the two ends of a sequence of interest, oligonucleotides are the best complementarity.
One of the primers recognizes complementarity, the sequence located upstream of the 5’-3’ strand of the DNA of interest. The same fragment of DNA with a sequence located upstream of the complementarity strand (3’-5’) is recognized by the other primer.
A primer is a single-stranded DNA, which when hybridized on sequences, allows selective replication. Their sizes are between 10-30 nucleotides.
This is how a polymerase chain reaction is performed. By setting appropriate temperatures in each stage, we can achieve multiple replicated DNA strands. The process is rapid and does not last longer than a few hours.
If your laboratory is short on the microbiology apparatus, you can buy them from Molecular Biology Products. We have PCR plates in different sizes and varying configurations, including PCR plates for ABI as well as 96 well-skirted plates.
- Kadri, K. (2019, June 7). Polymerase chain reaction (PCR): Principle and Applications. IntechOpen. Retrieved December 7, 2021, from https://www.intechopen.com/chapters/67558.