Some other enzymatic tools in rDNA technology

Polynucleotide Kinase:

• It is a phosphorylating enzyme that transfers the gamma phosphate of ATP to a dephosphorylated end of DNA or RNA.
• The enzyme is encoded by a gene of phage T4 and is extracted from E.coli cells infected with the phage. 
• This enzyme used after the Alkaline Phosphatase activity and introduces ³²P label (by using ATP).of DNA and RNA strands.
• Mg+2 and dithiothreitol are used in the reaction.

Terminal Deoxynucleotidyl Transferase

• The enzyme is a DNA polymerase that extend a strand without using a template.
• Any nucleotide that is provided in the reaction mixture is utilized to elongate the DNA strand.
• If only one kind of nucleotide is provided a mononucleotide polymer will be produced.

Alkaline Phosphatase

• The enzyme alkaline phosphatase removes the phosphate moiety at the 5’-end of DNA strand, whether it is part of blunt end single extension or a recessed end of a double-stranded DNA.
• The phosphate of RNA terminal is also removed by this enzyme.
• The commercial source of this enzymes is two sources: Bacterial and Calf intestinal phosphatases.

RNA dependent DNA polymerase:

• RNA dependent DNA polymerase is Reverse Transcriptase (RT).
• This enzyme synthesizes a single strand of DNA along an RNA template.
• It can also synthesize a second strand along the first one to make a ds complementary or cDNA.
• RT is usually utilized to copy mRNAs into ss or ds cDNA, and to make short labeled probes.

RNase H:

• RNase H is an endonuclease that is useful for degrading the RNA strand from a DNA:RNA hybrid molecule. It cut up the RNA into short fragments.