Molecular (gene) cloning involves extracting a target gene from DNA, inserting it into a vector, and using that vector to make multiple copies of the gene. The vector contains a marker gene and a restriction site that allows the target gene to be inserted. Restriction endonucleases cut the target DNA and vector DNA at specific restriction sites. The cut DNA fragments are then joined together using DNA ligase to form recombinant DNA, which is transferred into a host cell where it can be replicated through the cell's enzymatic machinery, producing many copies of the cloned gene. Molecular cloning has applications in gene study, production of human protein drugs, and vaccine preparation.

1. Molecular (Gene) Cloning
Mohammed Esawie, M.D., M.S.(c)

2. Background
• DNA amplification techniques produce
thousands or million copies of a particular
gene
• Either: In-vitro  PCR
In-vivo  Molecular Cloning
• In short: seek and find the gene  amplify it

4. Molecular (Gene) Cloning
• Definition??!
• Important components:
1- Target DNA
2- Restriction Endonucleases
3- Vectors
4- Marker gene
5- Recombinant DNA

5. On one side:
Target DNA & Restriction Endonucleases
• Secreted by bacteria  damage invading viral DNA
 Restricting its invasion
• Act on Restriction sites = palindromic Sequences
(4,6,8 bases read from 5’3’ in one DNA strand the
same as from 5’3’ in the antiparallel strand)
• EX:

6. On the other side:
Vector
• A molecule of DNA
• Contains:
Restriction site  will join to the gene to be
cloned
+ Marker gene 
to trace the vector after insertion
( to know which host cell accepts the vector) e.g
antibiotic resistance gene

7. On the other side:
Vector
• Types:
1- Prokaryotic Plasmids: extra-chromosomal
DNA (small, circular DNA.) e.g. E-coli plasmids
2- Viruses: naturally occurring viruses that infect
bacteria (Bacteriophage)
3- Cosmids: artificial recombinant plasmid can
insert larger DNA fragment up to 50 kb

8. Recombinant DNA
• Target DNA + Vector DNA
• Will be transferred from test tubes into host
cell that can provide the enzymatic machinery
for DNA replication (All DNA fragments
replication)

10. Molecular (Gene) Cloning Uses
• Gene study
• Synthesis of human protein drugs
• Vaccine preparation e.g. Hepatitis B Vaccine

11. Image Credits
• Amatakoon et al, DNA cloning and Restriction
Digestion. Pharmacognosy, 2007.
• Restriction enzymes & DNA ligase,
www.Khanacademy.org

12. Thank You