Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) is a molecular biological technique used to amplify or make
millions/billions of copies of a specific, small segment of DNA. The idea was developed by Kary Mullis
in 1983, it acts as a "molecular photocopier," allowing scientists to detect and study genetic material,
such as DNA from viruses, bacteria, or forensic samples. This tool has been widely used in various
molecular biological studies such as the diagnosis of pathogens (e.g., SARS-CoV-2), identification of
genetic disorders, forensic DNA profiling, and genetic engineering etc. Its significance lies in its
immense sensitivity and speed, enabling highly accuracy.
The Polymerase Chain Reaction (PCR) is a 3-step cyclic process which involves the basic three
steps such as denaturation, annealing and extension which allows to amplify the particular segment of
DNA. It is performed in a thermal cycler, the process repeats 25–35 times, heating and cooling to
separate strands, bind primers, and synthesize new DNA using Taq polymerase, creating millions of
copies.
Mechanism: The 3 Core Steps of PCR
1. Denaturation: The first step in PCR is denaturation. It involves Initial Denaturation, a longer
heating step (1–3 min) ensures all DNA is single-stranded followed by final denaturation to
separate the double-stranded DNA sample. It is done at 94-98 ℃ for 20-30 seconds. It breaks
the hydrogen bonds present between base pairs resulting the formation of single strands of
DNA.
2. Annealing: The second step is the annealing of the primer occurring typically at 50-65°C,
where DNA primers bind to complementary sequences on single-stranded template DNA. A
proper temperature needs to be maintained in order to allow highly specific and proper primer
hybridisation. This critical step sets the specificity of the reaction, enabling DNA polymerase
to start synthesis. The optimal temperature depends on primer length, GC content, and
sequence.
3. Extension: This is the final step in PCR cycle and a thermostable DNA polymerase is used for
this purpose. It is done at a temperature of 75-80 ℃ (72℃). Taq polymerase synthesizes a new
DNA strand and it adds nucleotides in the 5’-3’ direction and synthesises the complementary
strand of the DNA template.
References:
https://www.ncbi.nlm.nih.gov/books/NBK589663/
https://byjus.com/
Polymerase Chain Reaction (PCR) is a molecular biological technique used to amplify or make
millions/billions of copies of a specific, small segment of DNA. The idea was developed by Kary Mullis
in 1983, it acts as a "molecular photocopier," allowing scientists to detect and study genetic material,
such as DNA from viruses, bacteria, or forensic samples. This tool has been widely used in various
molecular biological studies such as the diagnosis of pathogens (e.g., SARS-CoV-2), identification of
genetic disorders, forensic DNA profiling, and genetic engineering etc. Its significance lies in its
immense sensitivity and speed, enabling highly accuracy.
The Polymerase Chain Reaction (PCR) is a 3-step cyclic process which involves the basic three
steps such as denaturation, annealing and extension which allows to amplify the particular segment of
DNA. It is performed in a thermal cycler, the process repeats 25–35 times, heating and cooling to
separate strands, bind primers, and synthesize new DNA using Taq polymerase, creating millions of
copies.
Mechanism: The 3 Core Steps of PCR
1. Denaturation: The first step in PCR is denaturation. It involves Initial Denaturation, a longer
heating step (1–3 min) ensures all DNA is single-stranded followed by final denaturation to
separate the double-stranded DNA sample. It is done at 94-98 ℃ for 20-30 seconds. It breaks
the hydrogen bonds present between base pairs resulting the formation of single strands of
DNA.
2. Annealing: The second step is the annealing of the primer occurring typically at 50-65°C,
where DNA primers bind to complementary sequences on single-stranded template DNA. A
proper temperature needs to be maintained in order to allow highly specific and proper primer
hybridisation. This critical step sets the specificity of the reaction, enabling DNA polymerase
to start synthesis. The optimal temperature depends on primer length, GC content, and
sequence.
3. Extension: This is the final step in PCR cycle and a thermostable DNA polymerase is used for
this purpose. It is done at a temperature of 75-80 ℃ (72℃). Taq polymerase synthesizes a new
DNA strand and it adds nucleotides in the 5’-3’ direction and synthesises the complementary
strand of the DNA template.
References:
https://www.ncbi.nlm.nih.gov/books/NBK589663/
https://byjus.com/
