Biomolecular Engineering Solutions for Renewable Specialty Chemicals. Группа авторов

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(primer) for the initiation of DNA strand synthesis. DNA polymerase‐I (DNA‐dependent DNA polymerase) is widely studied polymerase and has both polymerization and exonuclease activity that can help in synthesizing new strand as well as the degradation for proof reading or repair and primer removal. DNA polymerase I (or Pol I) takes part in the process of prokaryotic DNA replication. It was the first DNA polymerase discovered by Arthur Kornberg in 1956 (Lehman et al., 1958). Pol I has three different enzymatic activities: A 5′ →3′ DNA‐dependent DNA polymerase activity, a 3′ →5′ exonuclease activity that helps in proofreading, and a 5′ → 3′ exonuclease activity mediating nick translation during DNA repair. Pol I having polymerase but lacking nuclease activity is called klenow fragments (Klenow and Henningsen, 1970; Jacobsen et al., 1974).

      Taq DNA polymerase, a thermostable DNA polymerase isolated from Thermus aquaticus by Chien et al. (1976). It is frequently used in the polymerase chain reaction (PCR), to amplify small quantities of DNA. It has a functional 5′ → 3′ exonuclease domain at the N‐terminal, and 3′–5′ exonuclease domain was changed so it is not functional. Optimum temperature for Taq pol activity is 75–80 °C, with a half‐life of greater than 2 hours at 92.5 °C and minimum 9 minutes at 97.5 °C, and able to replicate a 1000 bp strand of DNA within 10 seconds at 72 °C.

      1.2.1.2 Nucleases

      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.

      1.2.1.3 Ligases

      Ligases are considered to be molecular glue in rDT, used to join two DNA segments. It also has role in various aspects of molecular biology such as replication, recombination, and cloning. In the presence of ligase enzyme when two DNA fragments are mixed under a certain condition, base pairing between two fragments occur which results in sealing of two different DNA fragments to make a chimera (Pascal et al., 2004). It occurs due to covalent bonds formation between 2′‐PO4 group and 3′‐OH group of adjustment strands.

       1.2.1.3.1 Mechanism of Action

      1.2.1.4 DNA‐modifying Enzymes

       1.2.1.4.1 Alkaline Phosphatase

      Alkaline phosphatase prevents self‐ligation of DNA molecule (vectors and gene of interest) in genetic engineering experiment by dephosphorylating phosphate group on 3′ end of the DNA molecule. It is extracted from E. coli or calf intestine. The enzymes catalyze the hydrolysis of monoesters in phosphoric acid which can moreover catalyze a trans‐phosphorylation reaction with large concentrations of phosphate acceptors. It can be used to prevent self‐ligation of vectors in the cloning experiments because alkaline phosphate‐treated DNA fragments lack the 5′‐phosphophate in the terminal, required for the actions of DNA ligases (Tamás et al., 2002).

       1.2.1.4.2 T4 Poly Nucleotide Kinase


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