1. Techniques used in Genetic Engineering
Dr.V. S. Bheemareddy

2. Following steps are involved in genetic engineering.
Isolation of desired gene or DNA.
Construction of recombinant DNA
Insertion of recombinant DNA in to host cells
Selection of transformed cells or organisms
Dr.V. S. Bheemareddy

3. 1. Restriction digestion
DNA is isolated from the source organism. It is subjected to digestion by various restriction enzymes. The specific REN cut DNA at specific palindromic sites.
Dr.V. S. Bheemareddy

4. This results in the formation of DNA fragments of different lengths.
Desired gene is present in one of such fragments. DNA fragments are subjected to gel electrophoresis to separate them from one another.
DNA fragment containing desired gene is isolated from the gel is used for gene cloning.
Dr.V. S. Bheemareddy

5. Dr.V. S. Bheemareddy

6. Dr.V. S. Bheemareddy

7. 2. Reverse transcription.
Every messenger RNA has information for protein synthesis in the form of triplet codons.
Messenger RNA coding for the synthesis of specific protein is isolated from the cell.
Reverse transcription is allowed by using reverse transcriptase enzyme.
Dr.V. S. Bheemareddy

8. Reverse transcriptase adds deoxyribonucleotides to 3’ OH group complementary to nucleotides of mRNA.
This results in the synthesis of single stranded DNA complementary to mRNA.
Dr.V. S. Bheemareddy

9. Dr.V. S. Bheemareddy

10. Oligonucleotides are bound together to form complete gene.
3. Chemical Synthesis
Small oligonucleotides are synthesized from free nucleotides by using free nucleotides. Gene machine is used for this purpose.
Oligonucleotides are bound together to form complete gene.
Dr.V. S. Bheemareddy

11. Construction of recombinant DNA
Recombinant DNA (rDNA) is hybrid DNA formed by the association the insertion of desired gene in the vector. It is also called as chimeric DNA. Recombinant DNA is constructed to achieve following goals.
To prevent desired gene from the lytic enzymes of host cell.
To allow the cloning of desired gene in host cell.
To enhance the expression of desired genes in host cells.
To facilitate the selection of recombinant host cells.
Dr.V. S. Bheemareddy

12. Dr.V. S. Bheemareddy

13. Insertion of recombinant DNA in to host cells
1.Transformation: Direct intake of recombinant DNA in to bacterial cells is called transformation.
The bacterial cells are placed in the 50mM CaCl2 Solution containing rDNA molecules. CaCl2 Solution facilitates the attachment of rDNA molecules to bacterial cells.
The solution is heated to 420 C and cooled. Due to sudden heating and cooling temporary pores are created in bacterial cell wall. Recombinant DNA molecules enter through pores produced in the cell wall.
Dr.V. S. Bheemareddy

14. Dr.V. S. Bheemareddy

15. Desired DNA is inserted in to Phage DNA to construct recombinant DNA.
2. Transduction
Insertion of foreign DNA in to bacterial cells through bacteriophage is called transduction. DNA of _phage is used for this purpose.
Desired DNA is inserted in to Phage DNA to construct recombinant DNA.
Proteins_of phage virus are synthesized and_placed around recombinant DNA to form derived phage.
These viral particles are allowed to infect bacteria maintained in culture. The recombinant DNA is inserted in to bacterial cell through infection.
Dr.V. S. Bheemareddy

16. Dr.V. S. Bheemareddy

17. 3. Electroporation
Electroporation is a method of inserting molecules in to cells by changing the permeability of membranes.
In genetic engineering this technique is used to introduce recombinant DNA in to bacterial cells, plant cells and animal cells.
Dr.V. S. Bheemareddy

18. Dr.V. S. Bheemareddy
Dr. V.S. Bheemareddy

19. The instrument used for this purpose is ultrasonicator.
Ultrasonication:
Method of inserting recombinant DNA in to host cells by causing membrane permeability changes through ultrasonic sound waves is called Ultrasonication.
The instrument used for this purpose is ultrasonicator.
Plant materials are exposed to ultrasonic sound waves , permeability of cell wall and cell membrane changed to permit the entry of recombinant DNA molecules in to host cells.
Ultrasonication is used to introduce recombinant DNA molecules in to seedlings, cotyledons, embryos and cell suspensions.
Dr.V. S. Bheemareddy

20. 5. Particle Bombardment Gun method
Shooting the DNA mixed tungsten or golden nanoparticles at plant and animal cells to incorporate recombinant DNA molecules is called particle bombardment or biolistics.
By this method recombinant DNA molecules could be introduced in to plant cells, animal cells
This method is used to transfer genes in to mammalian cells, organs, embryogenic cells and cells of callus.
Dr.V. S. Bheemareddy

21. Selection of transformed cells or organisms
One of the simplest genetic selection methods involves the use of antibiotics to select for the presence of vector molecules.
For example, the plasmid pBR322 contains genes for ampicillin (Apr) and tetracycline resistance (Tcr).
Only cells that have taken up the plasmid will be resistant, and these cells will therefore grow in the presence of the antibiotic.
Dr.V. S. Bheemareddy