1. The two scientists of USA, Stanley Cohen and Herbert Boyer (1972) isolated the antibiotic resistance gene by cutting the desired piece of DNA from the plasmid of the bacterium Salmonella typhimurium with the help of restriction enzymes (molecular scissors).
2. This piece of DNA was then linked with the plasmid DNA acting as vector by DNA ligase enzyme.
3. The newly formed recombinant DNA was transferred to bacterium Escherichia coli for replication by using the enzyme DNA polymerase. This process is called Cloning.
Recombinant DNA (rDNA): The hybrid DNA formed by combining DNA segment of two different organisms.
1. Cleaving enzymes
2. Lysing enzymes Micro-injection
3. Synthesizing enzymes
4. Joining enzymes
5. Alkaline phosphatases
(1) Cleaving Enzymes : These enzymes are used to break DNA molecules.
(a) Exonucleases : Cut off nucleotides from terminal ends of DNA
(b) Endonucleases : Make cut DNA at any point with in a DNA.
(c) Restriction Endonucleases : Make cut only speci®c position within a DNA. Single stranded free ends of DNA which can form hydrogen bonds with their complementary cut DNA segments are called 'Sticky Ends'. These ends can be joined by enzyme ligase.
(2) Lysing Enzymes : These enzymes are used to open the cells to get DNA.
For example : Lysozyme is used to dissolve the bacterial cell wall.
(3) Synthesizing :
(a) Reverse Transcriptases : Used in the synthesis of Complementary DNA strands on RNA templates.
(b) DNA Polymerases : Used in the synthesis of Complementary DNA strands on DNA templates.
(4) Joining Enzymes : Are used to join the cut ends of double stranded DNA (act as molecular glue). They join DNA fragments by forming phosphodiester bonds e.g., Ligase.
(5) Alkaline Phosphatases : These enzymes cut the phosphate group from the 5' end of linearised circular DNA to check its recircularization
