Biotechnology principles and process: Vectors, Restriction endonuclease enzymes, Tools of r-DNA technology

1. BIOTECHNOLOGY : PRINCIPLES AND
PROCESSES

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3. Herbert Boyer
• He performed studies on a couple of restriction enzymes
of the E. coli bacterium .
• Boyer observed that these enzymes have the capability
of cutting DNA strands in a particular fashion.
• which left what has became known as ‘sticky ends’ on
the strands.

4. Stanley Cohen.
• Cohen observed presence of plasmids in bacteria
• Plasmid: (autonomously replicating circular extra-
chromosomal DNA)

5. recombinant DNA
• The first r DNA constructed by Stanley Cohen and
Herbert Boyer in 1972.
• By linking a antibiotic resistance gene with a native
plasmid of Salmonella typhimurium.
• This r-DNA is transferred into Escherichia coli, a
bacterium closely related to Salmonella.

6. • Molecular scissors’– restriction enzymes.
• DNA ligases (molecular glue): "joins" the two
ends of DNA one having 3'OH and other
having 5‘P group.

7. recombinant DNA
EcoRI
EcoRI
Salmonella typhimurium

8. What is Biotechnology
• Biotechnology deals with techniques of using live
organisms or enzymes from organisms to produce
products and processes useful to humans.
• .

9. What is Biotechnology
curd, bread or wine, which are all microbe-
mediated processes.
in vitro fertilisation leading to a ‘test-tube’ baby.
Synthesising a gene,
 Developing a DNA vaccine
correcting a defective gene are all part of
biotechnology.

10. The European Federation of Biotechnology (EFB).
• The integration of natural science and organisms,
cells and molecular analogues for products and
services.
Molecular analogues: DNA,
RNA, Proteins and
Carbohydrates etc.

11. Modern biotechnology
Genetic
engineering
Maintenance of sterile ambience
(microbial contamination-free) in
chemical engineering processes
R-DNA

12. • Sexual reproduction: provides GENETIC
variations beneficial to the organism as well as
the population.
• Asexual reproduction: preserves the genetic
information.

13. • The techniques of genetic engineering which
include
• creation of recombinant DNA,
• gene cloning
• gene transfer.
Plasmid=Vector
Origin of replication (ori)
alien piece

14. Plasmid=Vector
Origin of replication (ori)
alien piece

15. TOOLS OF RECOMBINANT DNA TECHNOLOGY
• Restriction enzymes,
• Polymerase enzymes,
• Ligases,
• Vectors
• Host organism

16. Restriction enzymes
• Restriction enzymes belong to a larger class of
enzymes called nucleases.
• These are of two kinds;
• Exonucleases Endonucleases
.

17. • Two enzymes restricting the growth of
bacteriophage in Escherichia coli:
• Methyl transferase: added methyl groups to
E.coli DNA.
• Restriction endonuclease: cut bacteriophage
DNA.

18. Restriction Enzymes
• The first restriction endonuclease–Hind II.
• More than 900 restriction enzymes that have
been isolated from over 230 strains of bacteria

19. EcoRI
• EcoRI comes from Escherichia coli RY 13.
• Roman number : the order in which the
enzymes were isolated from that strain of
bacteria

20. • Each restriction endonuclease recognises a
specific palindromic nucleotide sequences in
the DNA.
• The length of palindromic nucleotide
sequences 6 base pairs.

21.  Each restriction endonuclease ‘inspecting’ the
length of a DNA sequence.
Finds its specific recognition sequence.
It will bind to the DNA
Cut each of the two strands of the double Helix at
specific points.

22. MALAYALAM”. a word-palindrome where the same
word is read in both directions,

23. Staggered
cut

24. • Even cut: Blunt ends: Flush ends

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

26. • DNA fragments are negatively charged molecules they
can be separated by forcing them to move towards the
anode under an electric field through a medium/matrix
• Matrix is agarose natural polymer sea weeds.
• Sieving effect provided by the agarose gel.

27. Cathode
end -
Anode end +

28. • the smaller the fragment size, the farther it moves.
• The separated DNA fragments can be visualised only
after staining the DNA with a compound known as
ethidium bromide followed by exposure to UV radiation.
• You can see bright orange coloured bands of DNA in a
ethidium bromide stained gel exposed to UV light

29. 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.

30. Cloning Vectors:
• Plasmids
• PBR 322 (developed by Boliver and Rodriguez )
• Bacteriophage genome
• Ti plasmid (Agrobacterium tumifaciens)
• Retrovirus genome

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

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

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

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

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