Gene cloning

By
D.Dolabza
V . s. lakshmi womens degree college
 What is gene cloning ?
The process of synthesizing multiple copies of a particular DNA sequence
using a bacteria cell or another organism as a host.
 GENE CLONING

TWO METHODS
1.) in vivo 2.) in vitro
 STEPS INVOLVED IN GENE CLONING
1.) Amplification of specific gene

2.) Cutting DNA at precise location

3.) Join two pieces of DNA

4.) Selection of small self replicating DNA

5.) Method to move a vector into a host

6.) Method to select host expressing recombinant DNA

 PLASMIDS – VEHICLES FOR CLONING

 Plasmids are naturally occurring Ampr

extrachromosomal DNA molecules. Ori

pBR322
 Plasmids are circular, double-stranded 4361bp

DNA. Tetr

 Plasmids are the means by which

antibiotic resistance is often transferred
from one bacteria to another.
 Plasmids can be cleaved by restriction LacZ
enzymes, leaving sticky or blunt ends. MCS

 Artificial plasmids can be constructed by pUC18

Ori
linking new DNA fragments to the sticky
Ampr
ends of plasmid.
 CLONING VECTORS Older cloning vector

 A cloning vector is a plasmid that can be Ampr

modified to carry new genes. Ori

pBR322
4361bp
 Plasmids useful as cloning vectors must have:
• An origin of replication. Tetr

• A selectable marker (antibiotic resistance

gene, such as amp1 and tetr).

Newer cloning vector
• Multiple cloning site (MCS) (site where

insertion of foreign DNA will not disrupt LacZ

MCS

replication or inactivate essential

pUC18
markers). Ori

• Easy to purify away from host DNA. Ampr

 Applications of gene cloning
Medical utility :-
Interferons Antibiotics

Vitamins Harmones
 Interferons
 Natural proteins produced by the cells of the immune system
of most vertebrates in response to challenges by foreign
agents such as viruses, parasites and tumor cells.

 Interferons belong to the large class of glycoproteins

known as cytokines.
Interferons assist the immune response by :
1. Inhibiting viral replication within host cells,

2.Activating natural killer cells and macrophage

3.Increasing antigen presentation to lymphocytes,

4.Inducing the resistance of host cells to viral infectio

Some recombinant DNA products being used in
human therapy
INSULIN for diabetics
HUMAN GROWTH HARMONES (HGH)

ERYTHROPOIETIN (EPO) for treating anemia

Several types of INTERFERONS

Many MONOCLONAL ANTIBODIES

 Agriculture utility :-

Bacillus thuringiensis bacteria produce a protein toxin that kills insect larvae
pests and is 80,000 times more toxic than the typical chemical insecticide.

Transgenic tomato, corn, potato, and cotton plants have been made that produce a
toxin from B. thuringiensis.
 Grains with improved nutritional characteristics:
Genes from bacteria and daffodil plants are transferred to rice using the
vector
Agrobacterium tumefaciens

Now a genetically modified strain of rice produces b-carotene, a

molecule that to vitamin A in animals.
is converted
 Attempts for impossible tasks

 Cleaning of :DDT

Industrial wastes

Reduce viscosity of crude oils

Conversion of sunlight to electricity

Degrade sulphur to coal

Bacterial fermentation to produce methane and other energy forms

Plastic degradation
 Biodegradable plastic-degrading enzyme from Pseudozyma antarcti

Pseudozyma antarctica JCM 10317 exhibits a strong degradation activity for biodegradable
plastics (BPs) such as agricultural mulch films composed of poly( butylene succinate) (PBS)
and poly(butylene succinate-co-adipate) (PBSA).

An enzyme named PaE was isolated and the gene encoding PaE was cloned from
the strain by functional complementation in Saccharomyces cerevisiae.
GLOWING CACTUS
 Risks of cloning

 >90% of cloning attempts fail to produce viable

offsprings
Cancers are caused

Higher rates of infections

Conclusion
 Thank you
For
Your Attention
A
PPT by
Dolabza