Download No Slide Title

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)
The Application of DNA
Recombinant Technology
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
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
Tools
Vector
Restriction and ligation enzymes
Host Cells
Vector
 Carriers move DNA from test tubes back into cells
 Pieces of DNA that can accept, carry, and replicate
other pieces of DNA
 An autonomously replicating genetic element used to
carry DNA fragments into a host for the purpose of
gene cloning
1. Bacterial plasmids
2. Bacteriophages (lambda phage)
3. Viruses
4. Yeast cells
Cloning vectors
Vector system
Host cell
Insert capacity (kb)
Plasmid
E. coli
0.1-10
Bacteriophage l
E. coli
10-20
Cosmid
E. coli
35-45
Bacteriophage P1
E. coli
80-100
BAC (bacterial artificial
chromosome)
E. coli
50-300
P1 bacteriophage-derived
AC
E. coli
100-300
YAC
Yeast
100-2,000
Cultured human cells
>2,000
Human AC
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
 Maximum size of insert is about 10 kb.
Lambda
It has a genome of about 50 kb of linear DNA
Only 37 to 52 kb DNA fragments can be
packaged into the lambda head.
Insertion vectors can hold up to 7 kb of cDNA.
Its life cycle is conducive to the use as a cloning
vector
The lytic cycle can be supported by only a
portion of the genes found in the lambda
genome.
Lambda
life cycle.
The lytic life
cycle produces
phage particles
immediately
The lysogenic
life cycle
requires genes
in the middle
of the
genome, which
can be
replaced
Lambda genome
Cosmid vectors
 Hybrid between a lambda vector and a plasmid.
 It can contain 33 to 45 kb.
Bacterial Artificial
chromosomes (BAC) vectors
These vectors are based on the E. coli F factor
These vectors are maintained at 1-2 copies per cell and
can hold > 300 kb of insert DNA.
Problems are low DNA yield from host cells.
Bacteriophage P1
These vectors are like lambda and can hold up to 110
to 115 kb of DNA .
This DNA can then be packaged by the P1 phage
protein coat.
The use of T4 in vitro packaging systems can enable
the recovery of 122 kb inserts
Yeast Artificial Chromosomes
Many DNA fragments cannot be propagated in
bacterial cells. Therefore yeast artificial
chromosomes can be built with a few specific
components.
1.Centromere
2.Telomere
3.Autonomously replicating sequence (ARS)
Genomic DNA is ligated between two telomeres
and the ligation products are transformed into yeast
cells
YAC cloning system
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
Vectors
Three important features
1. Cloning site
2. Ori-an origin of replication
3. A selectable marker
Coli Plasmid
pBR322
The plasmid pBR322 is one of the most commonly used E.coli cloning vectors. pBR322 is 4361 bp in
length and contains: (1) the replicon rep responsible for the replication of plasmid (source – plasmid
pMB1); (2) rop gene coding for the Rop protein, which promotes conversion of the unstable RNA I –
RNA II complex to a stable complex and serves to decrease copy number (source – plasmid pMB1); (3)
bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – transposon Tn3); (4) tet
gene, encoding tetracycline resistance protein (source – plasmid pSC101).
pUC18/19
pUC18 and pUC19 vectors are small, high copy number, E.coli plasmids, 2686 bp in length. They are identical except that
they contain multiple cloning sites (MCS) arranged in opposite orientations. pUC18/19 plasmids contain: (1) the pMB1
replicon rep responsible for the replication of plasmid (source – plasmid pBR322). The high copy number of pUC plasmids
is a result of the lack of the rop gene and a single point mutation in rep of pMB1; (2) bla gene, coding for beta-lactamase that
confers resistance to ampicillin (source – plasmid pBR322); (3) region of E.coli operon lac containing CAP protein binding
site, promoter Plac, lac repressor binding site and 5’-terminal part of the lacZ gene encoding the N-terminal fragment of
beta-galactosidase (source – M13mp18/19). This fragment, whose synthesis can be induced by IPTG, is capable of intraallelic (alfa) complementation with a defective form of beta-galactosidase encoded by host (mutation lacZDM15). In the
presence of IPTG, bacteria synthesize both fragments of the enzyme and form blue colonies on media with X-Gal.
Insertion of DNA into the MCS located within the lacZ gene (codons 6-7 of lacZ are replaced by MCS) inactivates the Nterminal fragment of beta-galactosidase and abolishes alfa-complementation. Bacteria carrying recombinant plasmids
therefore give rise to white colonies.
Agrobacterium tumefaciens
Genetic structure of the
Octopine Ti plasmid
Oncogenes
TL
Aux
Cyt
Opines
TR
Fig. 3
Binary vector system
Binary vector system
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
Selectable Marker Gene
Antibiotic
Description
Ampicillin (Amp)
Inhibits bacterial cell wall synthesis; inactivated by blactamase, which cleaves the b-lactam ring of amp
Kanamycin (Kan)
Binds to 30S ribosomal subunit and inhibits protein
synthesis; inactivated by a phosphotransferase
Neomycin (Neo)
Binds to 30S ribosomal subunit and inhibits protein
synthesis; inactivated by a phosphotransferase
Tetracycline (Tet)
Binds to 30S ribosomal subunit and inhibits protein
synthesis; tetr gene encodes a protein which prevents
transport of tet into the cell
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
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
E : Genus Escherichia
co: Species coli
R : Strain RY13
I : First endonuclease isolated
BamHI
B : Genus Bacillus
am: species amyloliquefaciens
H : Strain H
I : First endonuclease isolated
HindIII
H : Genus Haemophilus
in : species influenzae
d : strain Rd
III : Third endonuclease isolated
Specificity
Enzyme
Source
Sequence
End
BamHI
Bacillus amyloliquefaciens H
GGATCC
Sticky
BglII
Bacillus globigii
AGATCT
Sticky
EcoRI
Escherichia coli RY13
GAATTC
Sticky
EcoRII
Escherichia coli R245
CCTGG
Sticky
HaeIII
Haemophilus aegyptius
GGCC
Blunt
HindII
Haemophilus influenzae Rd
GTPyPuAC
Blunt
HindIII
Haemophilus influenzae Rd
AAGCTT
Sticky
HpaII
Haemophilus parainfluenzae
CCGG
Sticky
NotI
Nocardia otitidis-caviarum
GCGGCCGC Sticky
PstI
Providencia stuartii 164
CTGCAG
Sticky
Restriction Product
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
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