1. RECOMBINANT DNA TECHNOLOGY

2. DNA- the genetic secret!!
Encodes the genetic virusesinstructions of all known living organisms and many .
Nucleotides are the basic building block.
Nucleotide= Sugar + phosphate + Nitrogen bases.
4 Nitrogen bases
Anti-parallel strands

3. Nitrogen bases purines Adenine (A) Guanine (G) Thymine (T)
Cytosine( C)
found in pairs, with A & T and G & C Double helix
sequence and number of bases creates the diversity
DNA mRNA Proteins
 purines
 pyrimidines

4. What is Gene???
A gene is a stretch of DNA that codes for a type of protein that has a function in the organism.
It is a unit of heredity in a living organism.. All living things depend on genes
Genes hold the information to build and maintain an organism's cells and pass genetic traits to offspring.

5. Recombinant DNA Technology
Production of a unique DNA molecule by joining together two or more DNA fragments not normally associated with each other, which can replicate in the living cell.
Recombinant DNA is also called Chimeric DNA
Developed by Boyer and Cohen in 1973
3 different methods of DNA recombination
Transformation
Non-bacterial Transformation
Phage induction

7. Recombinant DNA Technology
Basic steps involved in recombinant DNA technology
Isolation of the gene of interest
Preparation of Vector DNA and DNA to be cloned
Insertion of the gene to the vector molecule and ligation
Introduction of the vector DNA to the appropriate host cell
Amplification of the recombinant DNA molecule in host cell.

8. Overview of rDNA technology
Bacterial cell
DNA containing gene of interest
Bacterial chromosome
Plasmid
Gene of interest
Isolate Plasmid
Enzymatically cleave DNA into fragments.
Isolate fragment with the
gene of interest.
Insert gene into plasmid.
Insert plasmid and gene into bacterium.
Culture bacteria.

9. Isolation of gene
DNA molecule is extracted from the cell by using cell lysing method
Homogenization
Centrifugation
Gene of interest is isolated using probes and electrophoresis
DNA which is to be cloned have to be inserted in to a vector
molecule which act as a carrier of the DNA to the host cell.
The choice of a vector depends on the design of the experimental system and how the cloned gene will be screened or utilized subsequently.
Commonly used vectors are Plasmid, bacteriophage, cosmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), yeast 2 micron plasmid, retrovirus, baculovirus vector

10. Plasmid vector (antibiotic resistance gene, such as ampr and tetr
Covalently closed, circular, double stranded DNA molecules that occur naturally and replicate extra chromosomally in bacteria and in some fungi.
Eg: pBR 322 and pUC-18
characteristic of an ideal plasmid
(i)Presence of minimum amount of its own DNA.
(ii) Recognition sites for restriction endonuclease
(iii)Presence of at least two markers with recognition site being present in one of the two markers
(iv)Relaxed replication control so that the recombinant plasmid is capable of forming several copies.
A plasmid containing resistance to an antibiotic (usually ampicillin) or Tetracycline, is used as a vector.
(antibiotic resistance gene, such as ampr and tetr

11. Restriction Endonucleases
Important tool for rDNA technology is the Restriction Enzymes
Bacterial enzymes that cut DNA molecules only at restriction sites
Molecular scissors
Palindromic sequences are the recognition sites
eg: EcoRI with recognition site GAATTC
5´ GAATTC 3´
3´ CTTAAG 5
Categorized into two groups based on type of cut
Cuts with sticky ends
Cuts with blunt ends
if one strand extends beyond the complementary region, then the DNA is said to possess an overhang and it will have sticky ends.

12. Commonly used restriction enzymes
EcoRI – Escherichia coli strain R, 1st enzyme
BamHI – Bacillus amyloliquefaciens strain H, 1st enzyme
DpnI – Diplococcus pneumoniae, 1st enzyme
HindIII – Haemophilus influenzae, strain D, 3rd enzyme
BglII – Bacillus globigii, 2nd enzyme
PstI – Providencia stuartii 164, 1st enzyme
Sau3AI – Staphylococcus aureus strain 3A, 1st enzyme
KpnI – Klebsiella pneumoniae, 1st enzyme

13. Restriction Endonucleases
Enzymes with staggered cuts  complementary ends
HindIII - leaves 5´ overhangs (“sticky”)
5’ --AAGCTT-- 3’ ’ --A AGCTT--3’
3’ --TTCGAA-- 5’ ’ –TTCGA A--5’
KpnI leaves 3´ overhangs (“sticky”)
5’--GGTACC-- 3’ ’ –GGTAC C-- 3’
3’--CCATGG-- 5’ ’ –C CATGG-- 5’
Enzymes that cut at same position on both
strands leave “blunt” ends
SmaI
5’ --CCCGGG-- 3’ 5’ --CCC GGG-- 3’
3’ --GGGCCC-- 5’ ’ --GGG CCC-- 5’

14. Actions of restriction enzymes-overview

15. Recombinant techniques
DNA to be cloned and the vector molecule are treated with the same restriction nuclease separately
It produces complimentary sticky ends
Sticky ends will self ligate through covalent bonding
This results in recombinant DNA molecule

16. Ligation of DNA
DNA Ligases close nicks in the phosphodiester backbone of DNA
DNA ligase is a enzyme that can link together DNA strands that have double-strand breaks (a break in both complementary strands of DNA).
Needs ATP
ATP

17. Cloning-Transformation
It is introduced into host cell by adding it into culture of plasmid free bacteria or animal cells.
Heating and adding calcium chloride favors the transformation
Once inside the host cell, the recombinant DNA begins to multiply and form the desired product.

18. Selection of recombinant cells

19. Selection of recombinant cells
Only bacteria which have taken up plasmid grow on ampicillin.
Blue-white selection:
white colonies have insert
blue colonies have no insert

20. Growing successfully….
The transformed cell are cultured and multiplied.
Colony of cell each containing the copy of the recombinant plasmid is obtained.

21. Non-Bacterial transformation
Microinjection, using micropipette.
The host cells are bombarded with high velocity micro-projectiles, such as particles of gold or tungsten that have been coated with DNA.

22. Phage Introduction Phage is used instead of bacteria.
In vitro packaging of a vector is used.
lambda or MI3 phages to produce phage plaques which contain recombinants.

23. Electroporation
It involves applying a brief (milliseconds) pulse high voltage electricity to create tiny holes in the bacterial cell wall that allows DNA to enter.

24. Applications… Gene therapy Medical diagnosis Xenotransplants
Pharmaceutical and Therapeutic Applications
Gene therapy
Medical diagnosis
Xenotransplants
Agricultural Applications
Production of transgenic organisms

25. Environmental applications
Many waste products of agriculture/industry do not break down naturally/break down slowly.
Many bacteria have been GE capable of breaking down oil and other organic wastes in Cheese making industry : GE Saccharomyces cerevisiae able to dispose of whey by converting lactose to alcohol.
Agricultural waste products, eg. corn husks, contain cellulose that normally decomposes slowly, can be converted into sugar by cellulase. Cellulase has been inserted in E.coli making it useful in waste management/disposal programs..

26. THANK YOU -PHARMA STREET