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Recombinant DNA Technology (Restriction endonuclease (also called…
Recombinant DNA Technology
What is rDNA?
Also known as a
DNA cloning
:<3:
Refers to
cutting DNA and pasting DNA
from different sources
Cloning- Reproduction of organism as a whole
without sexual fertilization
between male and female gametes
Gene cloning - insertion of foreign DNA(gene)
into another organism
Restriction endonuclease
also called
Restriction endonuclease (RE)
:fire:
Found in bacteria; as defense against bacteriophage infection
DNA cutting enzyme
:explode:
RE role
The act of cutting often called
digestion
:<3:
How? :star:
RE cuts the doubles strands of DNA by
breaking covalent bonds
between
:check: The
phosphate
of one covalent deoxyribosenucleotides
:check: The
sugar
of an adjacent deoxyribosenucleotides (phosphodiester bond)
Example of RE
Type of end
Double stranded ends
:fire: Blunt ends
Single stranded overhanging ends
Sticky ends or Cohesive ends
:fire: Sticky 3'
:fire: Sticky 5'
Sma1
EcoRI
Sac1
DNA Ligase
An enzyme
that join two DNA fragments
2 compatible sticky ends
2 blunt ends
Synthesize Phosphodiester bond
Common features between RE and DNA ligase
Both enzyme not
discriminate
the DNA
RE
can cut
any DNAs from any sources
DNA ligase
can joint together
any DNAs from any sources
DNA Cloning Vectors
Three important features:
has a site for insertion of
foreign
DNA
has a selectable
antibiotic resistant
marker
has the
ability to replicate autonomously
in bacterial host
Pieces of DNA that can accept, carry and replicate (clone) other pieces o DNA
DNA Plasmid
Screening of recombinants
Lac Z gene (β galactosidase)
White
colony screening
Colony carry recombinant plasmid
X-gal >
Blue
colony screening
Non-recombinant plasmid with intact Lac Z gene
Antibiotic resistant genes