What is a Polymerase?
Polymerase is an enzyme that is responsible for the formation of polymers of nucleotides such as nucleic acid.
The polymerase can be template-dependent or template-independent.
The polymerase that requires a template for its actions is known as template-dependent polymerase and the polymerase that does not depend on templates for the adding of nucleotides is known as a template-independent polymerase.
Template–dependent DNA polymerase requires a template for its functioning.
Template–dependent polymerase adds nucleotides to the 3’ end of primer which acts as a guide for this polymerase. eg: Terminal deoxynucleotidyltransferase.
Types of template-dependent polymerase
Template dependent polymerases are of two types:-
It is a polymerase that uses DNA as a template. It is also known as DNA-dependent DNA- polymerase. DNA polymerase also uses RNA as a template and it is known as RNA-dependent DNA polymerase or Reverse Transcriptase.
RNA polymerase also uses DNA as well as RNA as a template. The polymerase that uses DNA as the template is known as RNA polymerase or DNA-dependent RNA polymerase.
The polymerase that uses RNA as a template for the formation of the polynucleotide is known as RNA-dependent RNA polymerase orRdRp or RNA replicase.
Template independent polymerase
These polymerases do not require any template or primer for their activity. eg: Poly A polymerase.
Different types of Polymerase
The polymerase can be divided on the basis of two categories. They can be divided on the basis of function and structure.
Polymerase on the basis of function
There are different types of polymerase on the basis of their function:-
These are DNA- directed DNA polymerase, DdDP. There are different families under this category which is mentioned below with their example.
- Family A: DNA polymerase I; Pol ν, γ, θ.
- Family C: DNA polymerase III holoenzyme.
- Family X: Pol β, λ, μ. Terminal deoxynucleotidyltransferase (TDT).
- Family Y: DNA polymerase IV ( DinB) and DNA polymerase V ( UmuD’2C) [ These are the polymerases that are involved in SOS repair]. Pol η, ι, κ.
Reverse transcriptase (RT)
These are also known as the RNA- directed DNA polymerase (RdDP) because they depend on RNA template for the formation of DNA.
Telomerase- It is an enzyme that is responsible for synthesis of DNA at the telomericend of chromosome. It uses RNA as template.
DNA- directed RNA polymerase (DdRP, RNAP)
It is an RNA polymerase that uses DNA as the template for the formation of RNA.
- Multi-subunit (msDdRP) – RNA polymerase I, RNA polymerase II, RNA polymerase III.
- Single- subunit (ssDdRP) – T7 RNA polymerase, POLRMT.
- Primase, PrimPol.
These are The RNA polymerase that depends on RNA for its activity. It is also known as RdRP.
- Viral RNA replicase – It is a single unit replicase that is present in viruses.
- Eukaryotic cellular (cRdRP)- It is a dual unit replicase. It is found in eukaryotes.
Template- less RNA elongation
The enzymes that are involved in this process do not require a template.
- PAP, PNPase – These are the enzymes that are involved in the addition of poly-A Tail in messenger RNA during the modification of RNA.
Polymerases on the basis of structure
There are two types of superfamilies on the basis of structure. These are mentioned below:-
This is seen in all DNA polymerases and is also found in viral single–subunit polymerases. They have a conserved domain that is palm-shaped.
Double psi beta-barrel
It is seen in multi-subunit RNA polymerases, in cRdRP, “family D” DNA polymerases that are found in archaea.
It is a thermostable enzyme that is named after a thermophilic eubacterial microorganism Thermusaquaticus.
- It comes under the type DNA polymerase I.
- It is used in polymerase chain reaction (PCR).
- The microorganism from which it is isolated lives in hotsprings and hydrothermal vents.
- It can withstand high temperature that is provided during PCR so used in Polymerase chain reaction.
- The optimum temperature for this enzyme is 75-80.
- Half- life of this enzyme is 2 hours at 92.5 ˚C, 40 min at 95˚ C and 9 minutes at 97.5˚C.
- It can replicate 1000 base pair strand of DNA in approx. 10 sec at 72˚C.
- Deviation from optimal conditions will lead damage this enzyme.
- Potassium chloride and magnesium are also required for the activity of polymerase.
- The disadvantage of this enzyme is that it lacks 3’ to 5’ proofreading activity that results in low fidelity.
Polymerase Chain Reaction (PCR)
A polymerase chain reaction is a method that is used to make millions to billions of copies of DNA samples.
- The basic function of PCR is to amplify the DNA sample.
- It was invented in 1983 by Kary Mullis who was a biochemist.
- It is used in medical laboratory research for various biomedical research and criminal forensics.
- It is a method that is based on thermal cycling.
- The enzyme that is used in PCR is DNA Taq Polymerase which is heat stable.
Different stages of PCR
The different stages that are involved in Polymerase Chain Reaction (PCR) are:-
Initial Denaturation – It is the first step of Polymerase chain reaction (PCR). It is done at 94˚C for 30sec.
In this stage, dsDNA is denatured and it forms a single strand so that primer can attach to it and can promote elongation.
The second stage of polymerase chain reaction (PCR) consists of three steps:-
3 Steps of PCR cycle and Temperature
Denaturation in PCR – It is the step in which the left dsDNA is also denatured. It is also done at 94˚C for 30 sec.
Annealing – It is the step in which primer anneal to single-strand DNA. The appropriate temperature for annealing is 58˚C. The time period of this step is 30 sec.
Elongation – It is the step in which a complementary strand of the template is formed. The appropriate temperature and time for the elongation are 72˚C and 45 sec.
Final Elongation –It is the stage in which the final elongation of the dsDNA takes place. The optimum temperature and time for this stage are 72˚C and 3 min.
Final hold – It is the step at which the reaction is stopped. The temperature at which the final hold is done is 4˚C and it is for infinite time.
Applications of PCR (Polymerase Chain Reaction)
- This technique is used in DNA cloning for sequencing, gene cloning, and manipulation.
- It is used in the analysis of genetic fingerprints for DNA profiling.
- It is also used in the detection of pathogens in nucleic acid tests for the diagnosis of infectious diseases.
Components of PCR and their Functions
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