Recombinat DNA technology

1. Recombinant
DNA
TechnologyName : Heli Oza
Roll no. : 4
Class : HSGM sem 2

2. What is Recombinant DNA Technology ?
• Recombinant DNA technology or gene cloning or DNA cloning
• Creating same and multiple copies of same DNA or Gene again and again
using Vactors and Host organism.
What are the Application ?
• Recombinant human insulin
• Recombinant human growth hormone
• Recombinant hepatitis B vaccine
• Diagnosis of infection with HIV
• Crop improvement

3. • Golden rice
A recombinant variety of rice that has been engineered to express the enzymes responsible for β-
carotene biosynthesis.
• Herbicide-resistant crops
Commercial varieties of important agricultural crops (including soy, maize/corn, sorghum, canola,
alfalfa and cotton) have been developed that incorporate a recombinant gene that results in
resistance to the herbicide glyphosate (trade name Roundup), and simplifies weed control by
glyphosate application.
• Insect-resistant crops
Bacillus thuringeiensis is a bacterium that naturally produces a protein (Bt toxin) with insecticidal
properties.

4. Plasmid
DNA fragment
Recombinant
DNA
Host cell / Host organism

5. I. Identification & Isolation of Gene of interest or DNA fragment to be
cloned.
II. Insertion of this isolated gene in a suitable vector
III. Insertion of this isolated gene in a suitable vector (transformation)
IV. Screening
V. Multiplicatin or expression of the introduce gene in the host.
STAGES

6. I. Identification & Isolation of Gene of interest or
DNA fragment to be cloned.
Isolating DNA
Crude isolation of donor (foreign) DNA is
accomplished by isolating cells
1. disrupting lipid membranes with detergents .
2. destroying proteins with phenol or proteases
3. degrading RNAs with RNase
4. leaving DNA at the end
• Lysozyme – to break bacterial cell wall.
• Cellulase – to break plant cell wall.
• Chitinase – to break fungal cell wall.
• Ribonuclease – removes RNA.
• Protease – removes proteins (such as histones
that are associated with DNA).
Cutting DNA
1. DNA can be cut into large fragments by mechanical
shearing.
2. Restriction enzymes are the scissors of molecular
genetics.
Restriction enzymes (RE) are endonucleases that will
recognize specific nucleotide sequences in the DNA and
break the DNA chain at those points. (eg. EcoR1)
we have to cut both DNA and vector with same Restriction
enzyme.

7. Plasmid
DNA fragment
Restriction enzymes (RE) : EcoR1
CTTAAG
Target gene : Insulin
GAATTC

8. II. Insertion of this isolated gene in a suitable vector
Any DNA molecule that has the ability to replicate inside the host to which the desired gene has
integrated for cloninig
Vectors:
1. Plasmid
2. Bacteriophages
3. Yeast vector
4. cosmid

9. II. Insertion of this isolated gene in a suitable vector
Requirements for a cloning vector
a) Should be capable of replicating in host cell
b) Should have convenient RE sites for inserting DNA of interest
c) Should have a selectable marker to indicate which host cells received recombinant DNA molecule
d) Should be small and easy to isolate

10. Joining of DNA to Vactor
• Once you have isolated and cut the donor and vector DNAs, they must be joined together.
• The DNAs are mixed together in a tube. If both have been cut with the same RE.
• DNA ligase is the glue of molecular genetics
II. Insertion of this isolated gene in a suitable vector

11. Plasmid
DNA fragment
Recombinant
DNA
with the help of DNA ligase

12. III. Introduction of this vector into suitable organism
(transformation)
• In this step, the recombinant DNA is introduced into a recipient host cell. This process is
‘Transformation’. Bacterial cells do not accept foreign DNA easily. Therefore, they are treated to make
them ‘competent’ to accept new DNA.
• so, E.coli is treated with CaCl2 , the cells will take up the added DNA .
• Phase vectors are a more efficient introduction of DNA into bacteria. Phage vectors such as those
derived from bacteriophage l can carry larger inserts and can be introduced into bacteria more
efficiently. Recombinant bacteriophage can be introduction into E. coli by infection.

13. Plasmid
DNA fragment
Recombinant
DNA
Host cell / Host organism

14. IV. Screening Method
• Blue-White screening
• Insertional inactivation
• Genomic Library
V. Multiplicatin or expression of the introduce gene in the host.
The host cell will devide and replicates the cell.

15. Plasmid
DNA fragment
Recombinant
DNA
Host cell / Host organism

16. THANK YOU