S1 Nuclease
It is an endonuclease, which cleaves single-stranded DNA or single-strand protrusion of double-stranded DNA with cohesive ends. Because of S1 nuclease action, cohesive ends are converted into blunt ends. Thus, S1 nuclease is used to remove the incompatible ends.
DNA Pol-I, Klenow fragment
This fragment has the polymerase activity and 3′ → 5′ exonuclease activity of DNA Pol-I but does not have the 5′ → 3′ exonuclease activity. This 5′ → 3′ exonuclease activity is often troublesome because it degrades the 5′ terminus of primers that are bound to DNA templates and removes 5′-PO4 from the terminal DNA fragments that are to be used as substrates for ligation. The Klenow fragment can be used in the end filling and synthesis of DNA in cDNA clone.
Alkaline phosphatase
The cohesive ends of restriction enzyme treated plasmids instead of joining with the target DNA, sometimes reseal without taking the insert (target DNA) and are recircularized. To overcome this problem, the restricted vector (plasmid) is treated with the enzyme alkaline phosphatase, which removes the terminal 5′-PO4 group. The restriction fragments of the target DNA to be cloned are not treated with alkaline phosphatase. Therefore, the 5′end of the target DNA can covalently join with the 3′-end of the plasmid. Ligase action completes the formation of the rDNA.
Reverse transcriptase
Reverse transcriptase is used in the synthesis of cDNA using RNA template and also for the construction of cDNA clone bank.
Deoxyribonuclease I (DNAse I)
It is an endonuclease, which digests single- and double-stranded DNAs. This enzyme is useful for a variety of applications including nick translation, DNA foot printing, bisulphite-mediated mutagenesis and RNA purification. These enzymes have a role in genetic engineering and thus produce required specifications.