2-4.1.2 Application:

•  DNA ligase enzyme is used by cells to join the “okazaki fragments” during DNA replication process. In molecular cloning, ligase enzyme has been routinely used to construct a recombinant DNA. Followings are some of the examples of application of ligase enzyme in molecular cloning.Joining of adapters and linkers to blunt end DNA molecule.

•  Cloning of restricted DNA to vector to construct recombinant vector.

Fig 2-4.1.2: Ligation of a gene fragment into the vector and transformation of the cell.

2-4.2 Polynucleotide Kinase:

• PNK is a homotetramer with phosphatase activity at 3' end and kinase activity at 5' end with a tunnel like active site. The active site has side chains which interact with NTP donor's beta-phosphate and 3' phosphate of acceptor with an acid which activated 5' –OH. Lys-15 and Ser-16 are important for the kinase activity of the enzyme.
  
  
• The basic residues of active site of PNK interact with the negatively charged phosphates of the DNA.
  
  
• Polynucleotide kinase (PNK) catalyzes the transfer of a phosphate group (PO₄^-2) from γ position of ATP to the 5' end of either DNA or RNA and nucleoside monophosphate .
  
  
• PNK can convert 3' PO₄ /5' OH ends into 3' PO₄ /5' PO₄ ends which blocks further ligation by ligase enzyme.
  
  
• PNK is used to label the ends of DNA or RNA with radioactive phosphate group.
  
  
• T4 polynucleotide kinase is the most widely used PNK in molecular cloning experiments, which was isolated from T4 bacteriophage infected E.coli