ENZYME REQUIRED FOR RECOMBINANT DNA (rDNA) TECHNOLOGY

 Enzymes are used as biological tools in rDNA technology. They are discussed in detailed on following descriptions:

 F     Restriction Endonucleases(RE): There are two major types of restriction endonucleases: -----

·   Type I: Recognize a specific structure of nucleotides on the DNA molecule, but cut the DNA after 1000 – 5000 nucleotides from the recognized site. They cleave only one strand of DNA.

·   Type II: Recognize a particular target structure in a dsDNA & cleave the polynucleotide chain within/near the recognized sequence and provides DNA fragments of definite length and sequence.   

When dsDNA(double standed DNA) strands are cutted at the same place, the end is called as blunt end or whenever, the DNA to produce staggered ends in which single stranded short projections opposite and complementary to each other. The complementary arrangements are known as palindrome sequence or sticky end.   

RE	Source	Cleavage site
Bam HI	Bacillus amyloliquefaciens	5’ - G¯GA TC  C – 3’ 3’ – C C T AG­G – 5’
Eco RI	Escheria coli	5’ - G¯AA TC  T – 3’ 3’ – C  T T AG­A – 5’

  Function:

·        These enzymes cleave the DNA double strand.

·        Also used for:

1.       Isolating required genes.

2.       Mapping long strands of DNA.

3.       chromosome structure analyzing.

F     S1 Nuclease: Enzyme those cleaves ssDNA or single strand of dsDNA with cohesive ends. S1 nuclease causes conversion of cohesive ends into blunt ends. 

F     DNA ligases: This enzyme seals the single strand grooves/nicks in DNA which have 5^’®3^’ –OH termini. There are two extensively used DNA ligases. 

·   Ligase from E. coli: It can join the cohesive ends by utilizing NAD⁺ as coenzyme.

·   T₄ DNA ligase: It utilizes ATP when join blunt ends of the DNA fragments. 

*   Alkaline phosphatase: If the plasmid DNA is treated with RE, the cohesive ends of cut join together to form circular plasmid again. Alkaline phosphatase enzyme prevents the re-circularization of plasmid and helps in producing recombinant DNA molecules.   

*    Reverse Transcriptase: Which is nothing but a RNA dependent DNA polymerase enzyme. This produces ssDNA which is performed as template to produce complementary DNA chain. In recombinant DNA technology, this enzyme synthesizes cDNA from m–RNA which is collected from the human cell to represents the required gene. 

*    DNA Polymerases: are used in synthesize of cDNA template. They also catalyze a 5^’ ® 3^’ and 3^’ ® 5^’ exonucleolytic degradation of DNA. 

·  DNA Poly I

·  DNA Poly II

·  DNA Poly III  .ConstructedPlasmid