PCR also stands for polymerase chain reaction. This is a process of amplifying a known focused segment of genomic RNA or DNA. The process can also be used to amplify plasmid DNA in order to generate millions of RNA/DNA segment that have different uses in different sectors of molecular biology and other related fields.
When it comes to organisms, genomic DNA is referred to chromosomal DNA. However, researchers have also found dissimilarities between them. For example, plasmids are diagonal DNA segments in bacteria that are independent of the chromosomal DNA.
The process of PCR application is mainly found in the molecular biology for:
- Construction of DNA related phylogeny
- DNA cloning and sequencing
- Analysis of gene function
- Amplification of ancient DNA
- Diagnosis of hereditary diseases
- Genetic fingerprinting for forensic analysis etc.
Every PCR application includes heat stable DNA polymerase that is called Taq Polymerase. These types of bacteria live in hot springs & hydrothermal vents and the enzyme can stand conditions like protein denaturing. It is also really easier to use in PCR.
It is important for researchers to understand the fact that Primers have always been considered as the starting point for DNA synthesis. In addition, DNA polymerase can also be attached to a double stranded DNA. The process involves two different oligonucleotide DNA primers:
- The last sequence of the DNA strand that will be copied
- 20 to 40 bases long solo stranded DNA sequences that are ideally synthesized to accompany the beginning
The primers generally have 40 to 60 percent of rich content for better performance. Both forward primers and reverse primers are used to amplify and they are obligated to opposite strands of the DNA template with a compatible base pairing. After separation, the DNA strands into two different directions: 5' to the end and 3' to the end. So a researcher needs 2 primers to amplify each strand.
The Process of Polymerase Chain Reaction
The 3 stages of PCR are:
However, one will need 7 core ingredients for the setup. Mix the following reagents in a sterile microfuge tube:
- Genomic DNA template (around 50ng – 250ng)
- Reverse primer
- Forward primer
- Deionized water
- DNA nucleotide bases (dNTPs)
- Taq DNA polymerase
- Taq assay buffer (contains KCl, MgCl2, and Tris-HCl)
Denaturation: Heating process of the above ingredients for 3 to 5 minutes at a temperature of 94oC
Annealing: In this stage, the researcher needs to cool down the reaction for 10 to 30 seconds at a temperature of 50-65°C
Extension: Now, again heat the reaction for 5 to 10 minutes at a temperature of 72oC
By repeating the above-mentioned process for 30-40 cycles, researchers will be able to conduct a successful polymerase chain reaction. You have also come to know here, what it is and how it works. If you still have any question regarding this, then feel free to ask it by leaving us a mail. For buying total RNA of different species you can also consider visiting here: https://www.biochain.com/products/rna/total-rna-rna/. Time has come to sign off. Hope you have enjoyed reading it.