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BIOTECHNOLOGY PRINCIPLES AND PROCESSES
Terms and Definition

✓ Restriction enzyme: Protein that cuts

double-stranded DNA at a specific base sequence
✓ Recombinant DNA: Genetic material that has
been cut with restriction enzymes and spliced with
DNA from other organisms
✓ Transgenic organism: An individual with
recombinant DNA
✓ PCR (polymerase chain reaction): Amplifies DNA
in a test tube using the cell’s replication machinery
✓ Cloning: Creates an identical copy of an organism
✓ ORI: Origin of Replication
BIOTECHNOLOGY PRINCIPLES AND PROCESSES
Terms and Definition
✓ Elution: Extraction of separated fragments
of DNA from agarose gel electrophoresis
✓ Selectable markers: A gene or other
identifiable portion of DNA whose
inheritance can be followed and used in the
process of selection of transformed cells
from non transformed ones
✓ Insertional Inactivation: The process by
which a gene encoding a protein is
inactivated by the insertion of a foreign DNA
within the coding sequence of the protein
BIOTECHNOLOGY PRINCIPLES AND PROCESSES
Some Nick Names of Biotechnology
✓ Gene taxi: Plasmid
✓ Workhouse for gene cloning: pBR322 (an
artificial plasmid)
✓ Molecular scissor: Restriction ednoclusese
✓ Chemical Scalpel: Restriction ednoclusese
✓ Molecular glue: Ligases
✓ Chemical Knives: Restriction ednoclusese
✓ Natural genetic engineer: Agrobacterium
tumefaciens
✓ Mobile genetic element : Transposons
✓ Passenger DNA: Foregin DNA
BIOTECHNOLOGY PRINCIPLES AND PROCESSES
PROTEIN PRODUCTS OF RECOMBINANT DNA TECHNOLOGY
BIOTECHNOLOGY PRINCIPLES AND PROCESSES
Important Scientist
✓ W. Arber: In 1962 Discovered restriction
endonuclease in bacteria
✓ Har Gobind Khorana: In 1970 Synthesized
first artificial gene
✓ Arber, Smith and Nathan: Isolated first
"restriction endonuclease" from the
bacterium Haemophilus parainfluenzae
which can break DNA at specific sites
✓ Paul Berg: Started Genetic Engineering. He
was able to introduce a gene of SV-40 virus
into a bacterium with the help of lambda
phage. Paul Berg is "Father of genetic
engineering".
BIOTECHNOLOGY PRINCIPLES AND PROCESSES
Important Scientist
✓ Stanley Cohen and Herbert Boyer:
Generated first recombinant DNA molecule
by combining a gene from a bacterium with
plasmid of Escherichia coli.
✓ Boliver and Rodriguez: Developed the first
artificial cloning vector pBR322.
✓ Arber, Smith and Nathans: Awarded the
Nobel Prize for the discovery of restriction
endonuclease. These enzymes are called
"molecular scissors".
BIOTECHNOLOGY PRINCIPLES AND PROCESSES
Important Scientist
✓ Sanger: Received Nobel Prize in chemistry
twice— first time for structure of insulin (1954)
and second time (1980) for the base sequences
in DNA.
✓ Collins and Hons: Developed cosmid for the
first time
✓ Kary Mullis: Developed the Polymerase Chain
Reaction (PCR) technique and awarded Nobel
Prize in 1993.
BIOTECHNOLOGY PRINCIPLES AND PROCESSES
Important Scientist
✓ Lalji Singh: He developed a new technique of
DNA fingerprinting
✓ Alec Jeffreys: Developed DNA fingerprinting
technique.
✓ Ian Wilmut: Developed the World's first
mammalian clone named Dolly- a female
sheep.
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BIOTECHNOLOGY PRINCIPLES AND PROCESSES
Important Restriction endonucleases

Enzyme Organism Restriction Blunt or sticky

Sequence end

EcoRI Escherichia coli GAATTC Sticky

BamHI Bacillus GGATTC Sticky

amyloliquefaciens

Pvul Proteus vulgaris CGATCG Sticky

Pvu lI Proteus vulgaris CAGCTG Blunt

Hind Ill Haemophilus AAGCTT Sticky

influenzae

TaqI Thermus aquaticus TCGA Sticky

BIOTECHNOLOGY PRINCIPLES AND PROCESSES
Cloning Vector
✓ Plasmid: Size is 4361 bp, cloning limit is 0.1-10 kb.
Marker gene is ampicillin and the tetracycline
resistant gene. Example: PBR322 2.
✓ Bacterial Artificial Chromosome: Size is 11827,
cloning limit is 35-300 kb. Marker gene is
chloramphenicol and lactose metabolizing gene.
✓ It is a modification of f-plasmid and is artificially
synthesized. Example: pUvBBAC
BIOTECHNOLOGY PRINCIPLES AND PROCESSES
Cloning Vector
✓ Yeast Artificial Chromosome - Size of YAC is
11400 and
✓ They are used to clone DNA fragment of more
than 1Mb (106bp)
✓ Cosmid: Size of cosmid is 7900, cloning limit is
30-50 kb.
✓ Features similar to both phage and plasmid and
an example of it is super COS1
✓ Phagemid Vectors: Phagemid is a composite
structure made of bacteriophage and plasmid.
They are used for carrying larger DNA sequences.'