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Nucleases can cut or digest the DNA molecules either from one end or in middle by acting on phosphodiester bond, which forms the backbone of DNA (Nishino and Morikawa, 2002). Depending on the position of their digestion nucleases are of two types: exonucleases and endonucleases. Phosphodiester bonds present in the ends of the DNA are digested by exonucleases, removing nucleotides one at a time from either end. Whereas phosphodiester bonds present in the middle of the DNA strand are digested by endonucleases. Specificity of nucleases vary from source to source, Aspergillus oryzae’s endonuclease only cleaves single strands, whereas deoxyribonuclease I (DNase I), extracted from cow pancreas, cuts single as well as double‐stranded DNA molecules. DNase I not being sequence specific cuts DNA at random interior phosphodiester bond, leading to production of mononucleotides and very short oligonucleotides mixture. Some of the examples of nucleases are (i) Mung Bean Nuclease (isolated from mung bean sprouts) – a single‐strand‐specific DNA and RNA endonuclease which can degrade single strand overhangs from the end of DNA and RNA to make blunt ends. (ii) Nuclease S1 (isolated from Aspergillus sp.) – S1 nuclease is a single‐strand‐specific endonuclease that hydrolyzes single‐stranded RNA or DNA into 5′ mononucleotides. The enzyme will hydrolyze single‐stranded extensions in duplex DNA such as loops and gaps. S1 Nuclease is stable at 65 °C (Balabanova et al., 2012). (iii) Exonuclease III (isolated from E. coli) – removes single nucleotides from 3′ termini of the duplex DNA. It is generally used to make a set of nested deletions of the terminal of linear DNA strand. (iv) BAL31 nuclease (isolated from Alteromonas espejiana) – it is a 3′‐exonuclease and removes nucleotides from both 3′‐terminus of the two strands of linear DNA. (v) RNase H – it is an endonuclease that specifically hydrolyzes the phosphodiester bonds of RNA, when the RNA is hybridized to DNA (RNA‐DNA) (Cerritelli and Crouch, 2009). (vi) RNase P – the specificity of this enzyme is to cleave other RNA molecules at the junction of single‐stranded and the 5′ end of double‐stranded regions of RNA (Guerrier‐Takada et al., 1983).

Restriction endonuclease (RE) enzymes recognize and cleave the specific phosphodiester bond present in the DNA molecule (Smith and Welcox, 1970). Restriction enzymes are broadly classified into Type‐I, Type‐II, and Type‐III. For their functioning, they require specific temperature, ATP, and divalent magnesium ions. On digestion of the DNA molecule they can produce both blunt and sticky end. Type I REs interact with unmodified target site in dsDNA. They are bifunctional enzymes having methylase and endonuclease in a single protein molecule. They cleave DNA around 1000 bp away from the recognition site. For their function, both ATP and Mg²⁺ are required. Type II REs are highly specific and cleave within or very near to the recognition sequence due to this reason type II are used widely in genetic engineering. They do not require ATP for the restriction digestion, only Mg²⁺ is required. Type III REs cleave dsDNA at defined positions and need ATP, Mg²⁺. They cleave the DNA 24–26 bp away from the specific site.