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DNA Recombinant Technology
DNA recombinant
Genetic Engineering
The manipulation of an organism endowment by introducing
or eliminating specific gene
A gene of interest is inserted into another organism, enabling
it to be cloned, and thus studied more effectively
Design and construction of new combinations of genes (DNA)
New combinations/arrangements of DNA
DNA cloning
DNA Recombinant Technology
Technology used in the isolation or synthesis and joining together
of unlike pieces of DNA
These recombinant DNA molecules can then be introduced into
bacteria, yeasts, or other cells where they can replicate and
function (code for protein synthesis)
Overview of Genetic Engineering
 Gene of interest is isolated from appropriate organism
 Gene is recombined with a vector (carrier) DNA molecule
 Recombinant DNA is introduced into appropriate host cell
 Recombinant DNA is expressed at high levels in host cell
Gene product may be purified for use in treatments
(antibiotics, hormones, etc.)
Why
Detailed studies of the structure and function of a
gene at the molecular level require large quantities
of the individual gene in pure form
Tools
Restriction and ligation enzymes
Plasmid as a vector
Host Cells
Restriction Enzymes
Molecular scissors which isolated from bacteria where they are used as
Bacterial defense against viruses
Molecular scalpels to cut DNA in a precise and predictable manner
Enzyme produced by bacteria that typically recognize specific 4-8 base
pair sequences called restriction sites, and then cleave both DNA
strands at this site
A class of endo-nucleases that cleavage DNA after recognizing a
specific sequence
Members of the class of nucleases
Nuclease
Breaking the phosphodiester bonds that link adjacent
nucleotides in DNA and RNA molecules
 Endonuclease
Cleave nucleic acids at internal position
 Exonuclease
Progressively digest from the ends of the nucleic acid
molecules
Endonuclease
Type
Characteristics
I

II
III
Have both restriction and modification activity
 Cut at sites 1000 nucleotides or more away from recognition site
 ATP is required
 It has only restriction site activity
 Its cut is predictable and consistent manner at a site within or
adjacent to restriction site
 It require only magnesium ion as cofactor
Have both restriction and modification activity
Cut at sites closed to recognition site
ATP is required
Restriction Enzymes
 There are already more than 1200 type II enzymes isolated from prokaryotic
organism
 They recognize more than 130 different nucleotide sequence
 They scan a DNA molecule, stopping only when it recognizes a specific
sequence of nucleotides that are composed of symetrical, palindromic
sequence
Palindromic sequence:
The sequence read forward on one DNA strand is identical to the sequence
read in the opposite direction on the complementary strand
 To Avoid confusion, restriction endo-nucleases are named according to the
following nomenclature
Nomenclature
 The first letter is the initial letter of the genus name of the organism from
which the enzyme is isolated
 The second and third letters are usually the initial letters of the organisms
species name. It is written in italic
 A fourth letter, if any, indicates a particular strain organism
 Originally, roman numerals were meant to indicate the order in which
enzymes, isolated from the same organisms and strain, are eluted from a
chromatography column. More often, the roman numerals indicate the order
of discovery
Nomenclature
EcoRI
BamHI
HindIII
E : Genus Escherichia
co: Species coli
R : Strain RY13
I : First endonuclease isolated
B : Genus Bacillus
am: species amyloliquefaciens
H : Strain H
I : First endonuclease isolated
H : Genus Haemophilus
in : species influenzae
d : strain Rd
III : Third endonuclease isolated
Specificity
Enzyme
BamHI
BglII
EcoRI
EcoRII
HaeIII
HindII
HindIII
HpaII
NotI
PstI
Source
Bacillus amyloliquefaciens H
Bacillus globigii
Escherichia coli RY13
Escherichia coli R245
Haemophilus aegyptius
Haemophilus influenzae Rd
Haemophilus influenzae Rd
Haemophilus parainfluenzae
Nocardia otitidis-caviarum
Providencia stuartii 164
Sequence
GGATCC
AGATCT
GAATTC
CCTGG
GGCC
GTPyPuAC
AAGCTT
CCGG
GCGGCCGC
CTGCAG
End
Sticky
Sticky
Sticky
Sticky
Blunt
Blunt
Sticky
Sticky
Sticky
Sticky
Restriction enzymes
Restriction enzymes can be grouped by:
 number of nucleotides recognized (4, 6,8 base-cutters most common)
 kind of ends produced (5’ or 3’ overhang (cohesive=sticky), blunt=flush)
 degenerate or specific sequences
 whether cleavage occurs within the recognition sequence
Become familiar with the back of your molecular
biology catalog!
A restriction enzyme (EcoRI)
1. 6-base cutter
2. Specific palindromic
sequence (5’GAATTC)
3. Cuts within the
recognition sequence
(type II enzyme)
4. produces a 5’ overhang
(sticky end)
Restriction enzymes
Cloning
A collection of molecules or cells, all identical to an
original molecule or cell
 To "clone a gene" is to make many copies of it - for
example, in a population of bacteria
 Gene can be an exact copy of a natural gene
 Gene can be an altered version of a natural gene
 Recombinant DNA technology makes it possible
Vector
an autonomously replicating genetic element used to carry
DNA fragments into a host, typically E. coli, for the purpose
of gene cloning
• Plasmid vector
• Bacteriophage gamma vector
Plasmids
Naturally occurring extra-chromosomal DNA



Plasmids are circular double stranded DNA
Plasmids can be cleaved by restriction enzymes, leaving
sticky ends
Artificial plasmids can be constructed by linking new DNA
fragments to the sticky ends of plasmid
Cloning Vectors
Plasmids that can be modified to carry new genes

Plasmids useful as cloning vectors must have
• a replicator (origin of replication)
• a selectable marker (antibiotic resistance gene)
• a cloning site (site where insertion of foreign DNA will
not disrupt replication or inactivate essential markers
A typical plasmid vector with a
polylinker
Chimeric Plasmids
Named for mythological beasts with body parts from
several creatures



After cleavage of a plasmid with a restriction enzyme, a
foreign DNA fragment can be inserted
Ends of the plasmid/fragment are closed to form a
"recombinant plasmid"
Plasmid can replicate when placed in a suitable
bacterial host
Directional Cloning
Often one desires to insert foreign DNA in a
particular orientation



This can be done by making two cleavages with
two different restriction enzymes
Construct foreign DNA with same two restriction
enzymes
Foreign DNA can only be inserted in one
direction
Host Cells
Propagation of a DNA sequence must take place inside a living cell
(host cells)
Eschericia coli:
It provides a relatively simple and well understood genetic environment
The way to isolate plasmid is understood
It contains a single chromosome of approximately 5 Mbp
The genetic code is nearly universal
It replicates once every 22 minutes
It grows best with incubation at 37°Cin a culture medium that approximately
the nutrient available in the human digestive tract
Bacterial transformation
The cellular uptake and expression of DNA in a bacteria
Introduction of DNA into competent cell of bacteria
Requested element in transformation:
1. A suitable host organism in which to insert the gene
2. A self-replicating vector to carry the gene into the host organism
3. A means of selection for host cells that have taken up the gene
Selection of Transformant
A particularly important selective advantage offered by plasmid is
antibiotic resistance gene
It encodes for proteins that disable antibiotics secreted by
microorganism with which bacteria compete
Antibiotics function by several different mechanism
Antibiotics resistance:
A selectable marker that allows one to positively identify cells that have
been induced to take up plasmid DNA
Penicillin family (including ampicillin) interfere with cell wall biosynthesis
Kanamycin, tetracyclin, and chloramphenicol arrest bacterial cell growth by blocking
various steps in protein synthesis
Protein expression
- Gene is inserted into plasmid
- Plasmid is transformed into a host
cell (E. coli)
-Cell culture is prepared
-Each cell contains several copies of
the plasmid with gene
-Gene expression leads to the
production of protein
-Protein level may reach 30% of
total cellular protein
-Isolation of protein