Cloning Steps

1. Recombinant DNA
Technology.

3. • Recombinant DNA is a tool in understanding
the structure,
function , and
regulation of genes and their products.

4. The objective of Recombinant DNA
technology include ;
_Identifying genes.
_ Isolating genes .
_ Modifying genes .
_ Re-expressing genes in other hosts or
organisms .

5. Cloning Steps :
• Step 1 : DNA Isolation
_ Isolation of your gene of Interest .
.Step 2 : Recombinant DNA
_ Insertion of foreign DNA into bacterial plasmid
using restriction enzymes and DNA Ligase .
. Step 3 : Transformation
Insertion of recombinant DNA into bacteria by
making bacteria component .
. Step 4 : Production of recombinant DNA .

7. Enzymes
• Enzymes are proteins
- biological catalysts → help drive biochemical reactions.
• Enzymes names end with an ase (e.g.., endonuclease )
_ Bacteria have evolved a class of enzymes that destroy foreign DNA
(e.g.. Virus DNA).
Protect bacteria from bacteriophages (Virus).
_ Bacteriophages cannot multiply if their DNA is destroyed by host .

8. Restriction Endonucleases
• Restriction endonucleases RESTRICT viruses
- viral genome is destroyed upon entry.
• Restriction endonucleases = Restriction enzymes
- Endo (inside), nuclease (cuts nucleic acid)
• Restriction endonucleases recognizes a short and
specific DNA sequence and cuts it from insides.
• The specific DNA sequence is called recognition
sequence.

9. Bacteriophage Life Graph
• Animation of the Lytic Life cycle of a
Bacteriophage

10. Nomenclature
• Smith and Nathans (1973) proposed enzymes naming
scheme .
- three-letter acronym for each enzyme derived from
the source organism
- Frist letter fro genus
- Next two letters represent species
- Additional latter or number represent the strain or
serotypes
• For example: The enzyme Hindll was isolated from
Haemophilus influenzae serotyped.