Agarose gel electrophoresis, isolation of DNA and PCR reaction, Transformation methods of DNA in the host, downstream processing

1. Separation and isolation of DNA
fragments
• DNA fragments can be separated by a technique
known as agarose gel electrophoresis.

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3. Cathode
end -
Anode end +
• Matrix----------agarose----natural polymer ------- sea weeds.
• Sieving effect provided by the agarose gel.

5. • ethidium bromide followed by exposure to UV
radiation.
• bright orange coloured bands of DNA

6. Elution.
• The separated bands of DNA are cut out from
the agarose gel and extracted from the gel
piece. This step is known as elution.

7. Cloning Vectors:
• Plasmids
• PBR 322 (developed by Boliver and Rodriguez )
• Bacteriophage genome
• Ti plasmid (Agrobacterium tumifaciens)
• Retrovirus genome (RNA)
pUC19 is one of a series of plasmid cloning vectors

8. Other examples for vectors
• Cosmid:
• A cosmid is a type of hybrid plasmid that contains
a Lambda phage cos sequence and plasmid
Cosmids (cos sites + plasmid = cosmids).
• YAC
• BAC
• MAC

10. • 1000 bp = 1 kb
• 1 million bp= 1000 kb = 1 megabase pairs.

11. PBR 322
Origin of replication (ori) :
Selectable marker :
Cloning sites:

12. PBR 322
rop GENE: codes for the
proteins involved in the
replication of the
plasmid

13. Selective markers:
• Two selective markers
present in pBR322:
• Tetracycline resistant
gene and Ampicillin
resistance gene.

14. Cloning sites/recognition sites
• Total eight cloning
sites present in
PBR322 for eight
different restriction
endonuclease
enzymes.

15. Transformed cells Non Transformed cells
E.coli E.coli
Ampicillin
Ampicillin
Tetracyclin

16. Alternative selectable markers
Lac Z gene
• On the basis of their ability to produce colour
in the presence of a chromogenic substrate.
• Lac z gene  B-galactosidase  X-gal (colour
less)  Blue colour..

17. Lac Z
gene

19. Insertional inactivation.
• . In this, a recombinant DNA is inserted within
the coding sequence of an enzyme, β-
galactosidase.
• This results into inactivation of the enzyme,
which is referred to as insertional inactivation.

21. Cloning vector for Plants
Ti plasmid
• Ti plasmid Agrobacterium tumifaciens  dicot plants.
• T-DNA’ to transform normal plant cells into a tumor.

22. Cloning vector for animals
Retrovial genome (RNA)
Retroviruses in animals have the ability to
transform normal cells into cancerous cells.

23. Competent Host (For Transformation with
Recombinant DNA)
• Calcium chloride method (bacteria)
• Heat shock method (42°C ) (Bacterai)
• Microinjection method (Animals)
• Gene gun method (Plants).
• Disarmed pathogen’ vectors.

24. Calcium chloride method
for bacteria
divalent cation, such as calcium,

25. Heat shock method

26. Microinjection method (Animals)

27. Microinjection method (Animals)

28. Gene gun method (Plants)
• Bombarded or Biolistics method:
• Plant cells Bombarded with high velocity micro-
particles of gold or tungsten coated with DNA in a
method known as biolistics or gene gun.

29. Gene gun method (Plants)
gold or tungsten
coated with DNA

31. Processes of recombinant DNA
technology
I.Isolation of DNA.
• In order to cut the DNA with restriction
enzymes, it needs to be in pure form, free from
other macro-molecules.
• RNA, proteins, polysaccharides and also lipids

32. Isolation of DNA by treating the bacterial cells/plant
or animal tissue with enzymes
• lysozyme (bacteria),
• cellulase (plant cells),
• chitinase (fungus).
• Detergents used for lysis cell membrane.

33. • RNA  ribonuclease (RNAse)
• proteins  protease.
• DNA ultimately precipitates out
after the addition of chilled
ethanol.
• This can be seen as collection of
fine threads in the suspension…

35. Polymerase Chain Reaction (PCR)
• Amplification of Gene of Interest using
PCR.
• 1.Denaturation: 94°C
• 2. Primer annealing; (40-60°C)
• 3.Extension of primers (72°C)

36. Annealing
• Two sets of primers (DNA Primers) (small
chemically synthesised oligonucleotides (10-18
nucleotides).

37. Extension of primers (72°C):
• Taq DNA polymerase enzyme
• thermostable DNA polymerase (isolated from a
bacterium, Thermus aquaticus).
• 30 cycles: 1 billion copies.

39. Two sets
of DNA
Primers:

40. Processes of recombinant DNA
technology

41. Transformed cells Non Transformed cells
E.coli E.coli
Ampicillin
Ampicillin
Tetracyclin

42. Insertion of Recombinant DNA into the Host
Cell/Organism
AMP
Plasmid with Ampicillin
resistance gene
Inserted
gene
E.COLI
Recombinent
E.coli
resistance to
Ampicilin
antibiotic

43. if a recombinant DNA bearing gene for resistance to an
antibiotic (e.g., ampicillin) is transferred into E. coli
cells..
• the host cells become transformed into ampicillin-
resistant cells.
• If we spread the transformed cells on agar plates
containing ampicillin, only transformants will grow,
untransformed recipient cells will die.

44. Obtaining the Foreign Gene Product
• If any protein
encoding gene
is expressed in a
heterologous
host, it is called
a recombinant
protein. E.coli
Insulin
gene
R-DNA
Insulin
protein

45. • The recombinant cells may be grown on a small
scale in the laboratory.
• The cultures may be used for extracting the
desired protein..

46. • The cells can also be multiplied in a continuous
culture system .
Fresh
medium
used
medium is
drained out

47. • The cells are in physiologically most active
log/exponential phase.
• This type of culturing method produces a larger
biomass leading to higher yields of desired
protein.

48. Bioreactors
• Bioreactors volumes (100-1000 litres) of culture
can be processed, was required.
• In bioreactors raw materials biologically
converted into specific products.
• Bioreactors providing optimum growth
conditions (temperature, pH, substrate, salts,
vitamins, oxygen)….

49. • The most commonly used bioreactors are of
stirring type….
• A stirred-tank reactor is usually cylindrical or
with a curved base to facilitate the mixing of the
reactor contents…

51. Downstream Processing
• The processes include separation and
purification, which are collectively referred to as
downstream processing.

52. Product
Formulation
Quality control
testing
it is ready for marketing…
DRUG
Undergo clinical
trainls
separation and purification

54. Dr. HarinathaReddy Aswartha
Assistant professor
Department of Microbiology
biohari14@gmail.com