Molecular Cloning Methods
Gene cloning (DNA cloning)
• Clone: a group of identical cells or
organisms.
• Gene cloning: generating many copies of
a gene by inserting it into an organism,
such as a bacterium, where it can
replicate along with the host.
• A gene cloning experiment is to
place a foreign gene into bacterial
cells, isolate individual cells, and
grow colonies from each of them.
All the cells in each colony are
identical and will contain the foreign
gene.
DNA cloning
procedure
(RE)
(Ligase)
transformation
selection
Restriction
endonucleases
The first restriction 44=256bp
enzyme was found in
E.coli in the late 1960s.
46=4096bp
HindII
:Haemophilus influenzae
strain Rd
Present frequency: 4n
n = number of restriction
enzyme recognition sequence
isoschizomers
48=~65000bp
Sticky end and blunt end
Sticky end
Restriction enzymes make staggered cuts in the two DNA
strands, leaving single-stranded overhangs.
EcoRI 3’ recession
5’---GAATTC--- 3’ 5’---G3’ 5’AATTC---
+
3’---CTTAAG--- 5’ 3’---CTTAA5’ 3’G---
5’ protruding
PstI
5’---CTGCAG---3’ 5’---CTGCA3’ + 5’ G---
3’---GACGTC---5’ 3’---G5’ 3’ACGTC---
Blunt end
Restriction enzymes cut in the middle of its sequence,
produce no overhangs.
SmaI
5’---CCCGGG---3’ 5’---CCC3’ 5’GGG---3’
+
3’---GGGCCC---5’ 3’---GGG5’ 3’CCC---5’
Sticky end
Blunt end
Palindromes :
sequences with twofold symmetry are
called palindromes.
5’---GAATTC---3’
3’---CTTAAG---5’
Why do restriction
enzymes not
destroy the host
cell’s own DNA?
Restriction-modification
system (R-M system)
Almost all restriction
enzymes are paired
with methylases.
After methylation,
DNA sites are
protected against most
restriction enzymes.
Digestion and ligation
5
ATP
Joining of vector to insert
DNA ligase ligase (3)
+ ATP
(1)
ligase-AMP
(2)
All gene cloning
experiments
require such
carrier , which
we call vectors
to allow
replication of
recombinant
DNA.
Plasmids, small, pBR322
circular DNAs
that are
independent of
the host
chromosome
DNA cloning
procedure
(RE)
(Ligase)
transformation
selection
Vector typically have three
characteristics
They contain an origin of replication that
allows them to replicate independently of the
chromosome of the host.
They contain a selectable marker that allows
cells that contain the vector (and any attached
DNA) to be readily identified.
They have single sites for one or more
restriction enzymes. This allows DNA fragments
to be inserted at a defined point within an
otherwise intact vector.
• vector
– Plasmid
– Phage as a plasmid
– Cosmid
– M13 phage
– Phagemid
– Expression vector
Cloning foreign DNA using the PstI site of
pBR322
Step.1
Step.3
Step.2
•Electroporation: Electrical treatment of cells
that induces transient pores, through which DNA
is taken into the cell.
Screening bacteria by replica plating
Positive selection Negative selection
pUC
MCS = multiple cloning site
-peptide of -galactosidase
pUC Plasmid of University of
California
4.2. Blue/White Color Screening
4.2. Blue/White Color Screening
Increase efficient of
cloning
(1) Improve screening
(2) Prevent vector self-
ligation
a. Dephosphorylation of
DNA 5’-phosphate
b.Using two different
restriction enzymes.
Gateway Cloning
• Gateway cloning, relies on lambda
bacteriophage biology because these
vectors use the integration and excision
sites from the lambda genome for adding
an insert.
• Moving the insert from the entry vector to a
destination vector uses the LR cloning reaction.
• The BP cloning reaction removes the gene of
interest from the destination vector and put it back
into the entry vector.
CcdB, a Toxin That Kills Bacteria Without an
Insert
• This natural system to kill the bacteria that
do not have the insert in the vector after
cloning and transformation.
Phage as a vectors
Bacteriophage is nature vectors that
transduce bacterial DNA form one cell to
another.
Phage vector have a nature advantage
over plasmids:
They infect cells much more efficiently
than plasmids transform cells, so the
yield of clones with phage vectors is
usually higher.
With phage vectors, clones are not
colonies of cells, but plaques formed when
a phage clears out a hole in a lawn of
bacteria.
First phage vector:
Charon phages: Blattner took out the
region of middle of phage DNA, but
retained the genes needed for phage
replication.
Advantage of phages:
They can accommodate much more foreign DNA
-about 20 kb
-use for genomic library
some phage have the advantage of a minimum
size requirement
- at least 12kb
- library needs about 500,000 clones for cover
whole human genomic DNA (DNA size locate
between 12Kb to 20kb)
EcoRI present
frequency ~ 24 =
4kb is small 12kb.
Increase size of
DNA fragment
Partial
digestion
Use mechanical
means such
ultra-sound
M13 phage vectors
Contain -
galactosidase gene
fragment and multiple
clone site
Advantage: single-
stranded DNA, ease to
perform side direct
mutagenesis.
Q: How to get single strand DNA?
Phagemids
There are like the cosmids in that they have
characteristics of both phages and plasmids.
The popular one is pBluescript (pBS)
pBS
• vector
– Plasmid
– Phage as a plasmid
– Cosmid
– M13 phage
– Phagemid
• Target DNA (foreign DNA)
– PCR
– cDNA
• cDNA library
• RT-PCR
• Identifying a specific clone
– Specific probe
Polymerase Chain Reaction
A technique for amplifying a specific
segment of DNA by using a thermostable
DNA polymerase, deoxyribonucleotides, and
primer sequences in mutliple cycles of
denatureation, renaturation, and DNA
synthesis. Also Called PCR.
DNA replication
• DNA temple, primer, dNTP and DNA
polymerase
PCR
Using RT-PCR to clone a single cDNA
cDNA: A DNA copy of an RNA, made by
reverse transcription.
TA Cloning of Polymerase Chain Reaction (PCR)
Products
Expression vectors
• Goal: make a large quantity of the
gene’s product.
• Prokaryotic expression vector (a
promoter and ribosome binding site are
required)
– Promoter
• Inducible expression vector
– Fusion protein
• Eukaryotic expression vector
Promoter:
strong promoter (constitute)
trp, T3 and T7 promoter
inducible promoter
lac promoter
strong
promoter
(constitute)
Inducible expression vector
Keep a cloned gene repressed until it is time to
express it.
Reason: toxic to bacteria, interfere
bacterial growth.
lac promoter:
Inducer : IPTG
lacI system:
lacI as a repressor
Inducer: IPTG
lac and T7 RNA
polymerase
IPTG lac promoter
T7 RNA polymerase
T7 promoter gene
expression
Most expression vectors produce fusion
proteins
oligo-His (6X)
GST (Glutathione S transferase)
A case of fusion protein
Using an oligo-
histidine
expression
vector
How to do
construction?
How to do
expression and
purification
Phage expression
vector
for expression
library
vector: lgt11
Promoter: lac
operon (lacZ)
Mammalian Expression Vectors
Electroporation
Microinjection
Transformation of Eukaryotic
•Lipofection: Delivery into eukaryotic cell
DNA, RNA, or other compounds that
have been encapsulated in an artificial
phospholipid vesicle.
•Liposome: A circular collection of lipid
molecules in which the hydrophobic
proteins of the molecule are facing
inward; a lipid vesicle with an aqueous
interior that can carry nucleic acids,
drugs, or other therapeutic agents.
Microinjection: The introduction of DNA or other
compounds into a single eukaryotic cell with a fine,
microscopic needle.
Expression a gene in a baculovirus
genome: 130kb circular DNA
Major viral structural protein: polyhedrin
Up to 10% of the dry mass of the dead
insect which infected baculovirus is this one
protein.
Application: replace cloned gene under the
control of the polyhedrin promoter.
Expression Vector for plant
regular expression vector
Ti-plasmid
Agrobacterium
Using a T-
DNA plasmid
to introduce a
gene into
tobacco plants
•Biolistics: Delivery of DNA to plant and animal cells and
organelles by means of DNA-coated pellets that are
fired under pressure at high speed. Also called
microprojectile bombardment
A DNA
Library Is a
Collection
of Genes
or phages
Making a cDNA library
cDNA: A DNA copy of an RNA, made by
reverse transcription.
(TdT)
(plasmid or
phage e.g
gt11)
or Random primer
