The following points highlight the five main enzymes that are to be used in DNA. The enzymes are: 1. Phosphatases 2. Taq Polymerase 3. Nucleases 4. Reverse Transcriptase 5. DNA Ligating Enzyme.
Enzyme # 1. Phosphatases:
Phosphatases (Alkaline) are glycoproteins with two identical subunits that hydrolytically remove phosphate groups from DNA molecule. Alkaline phosphatases are used to reduce unfeasible ligation of cleaved plasmids in recombinant process i.e., dephosphorylation of vector DNA will prevent recircularization of vector in cloning process. This enzyme is also used for mapping and finger printing studies.
Enzyme # 2. Taq Polymerase:
Taq polymerases are DNA dependent DNA polymerase from Thermus aquaticus, primarily used for synthesis of longer stretches of DNA. Thermus aquaticus is the source for this enzyme which is an extreme thermophile, living in hot springs.
Due to its thermophillic habitats, it shows extreme resistant to high temperature, which exhibits peak of activity at temperature optimum of 72°C. Taq polymerase consists of single polypeptide chain with a molecular weight of 90,000 daltons.
This enzyme is particularly useful when reactions need to take place at high temperature, denaturation of high G + C content into DNA. Thermostable property of this enzyme is significant in the polymerase chain reaction, where it is used to extend primers in the process of repeated rounds of heating, cooling and complete the synthesis of new DNA strand.
Enzyme # 3. Nucleases:
Nucleases are DNases and RNases. DNases digest both the strands of DNA. It can hydrolyse each DNA strand independently in presence of Mg+ ion. DNases are used in the purification of DNA by eliminating contaminated DNA. It is also widely used in foot printing and nick translation.
RNases cleaves phosphodiester bond between the two adjacent nucleotides. RNase cleaves the bond next to uracil and guanine. Aspergillus and bovine pancreas are the chief source for RNase. The RNase H is widely employed in cDNA preparation where it removes mRNA from RNA-DNA hybrid. Removal of polyA tail requires RNase.
Enzyme # 4. Reverse Transcriptase (RTase):
These are RNA-dependent DNA polymerases. They are commonly called as reverse transcriptases—capable of reversing the flow of information by transcription in the ‘central dogma’. They are encoded by retroviruses. It consists of two important subunits, requires DNA complementary to RNA template. Like other DNA polymerases, they direct DNA synthesis in a 5′-3′ direction.
This enzyme for commercial use is chiefly obtained from avian myeloblast virus. Most important use of RTase in genetic engineering experiment is in the synthesis of DNA from RNA when making cDNA libraries. They can also be used in sequencing of RNA or DNA. RTase shows high rate of error as they do not have proof reading 3′-5′ exonuclease activity.
Enzyme # 5. DNA Ligating Enzyme (Ligases):
Ligases acts as a key player in genetic engineering experiment for its role as molecular suture, where it facilitates joining of DNA fragments. The main source of this enzyme is the T4 phage virus. The two DNA pieces are efficiently joined by DNA ligase.
The joining of DNA fragments requires ATP for T4 DNA ligase or NAD+ in the case of E. coli ligase. Joining of DNA fragments is accomplished by forming a covalent bond between the 5′ phosphoryl of one strand and 3′ hydroxyl of the adjacent strand.
Thus, it catalyses the end to end joining of DNA duplex at the base paired end. During sealing process, two phosphodiester bonds are formed by T4 DNA ligase. The stability of joined fragments is due to the formation of 3′ to 5′ phosphoto diester linkage between cohesive ends.
The blunt ends produced by certain restriction enzymes may hinder the sealing process (Fig. 13.1). Hence, it is estimated to use ligase at high strength to accomplish sealing process.
