Download Cloning

Cloning
Guanqun Yuan
12-13-2004
R.A.Scott Group Meeting
Outline
 What is cloning?
 How to do cloning?
 Validation
 Application
What is Cloning?





Generating identical copies of organisms, cells,
or replicating nucleic acid sequences from
organisms
involving human intervention.
Giving rise to new generation
Dolly (the sheep) is a clone, but a natural
identical twin is not a clone.
DNA sequence amplified by growth is a clone,
Identical DNA molecules produced in vitro (a
PCR rxn) is not a clone.
Outline
 What is cloning?
 How to do cloning?
 Validation
 Application
How?
ORI
Cells that do
not take up
plasmids die on
ampicillin plates
Amp R.
ORI
Plasmid vector
+
DNA fragment
to be cloned
Enzymatically
insert DNA into
plasmid vector
Amp R.
Mix E.coli cells with
plasmids
in presence of
Recombinant
CaCl2 Culture on nutrient
plasmid
agar plates containing
ampicillin
Transformed
E.coli cell
survives
Bacterial
chromosome
Independent
plasmid
replication
Cell
multiplication
Composition

Vectors
Plasmids, or phage

Plasmid vector
The cell
E.coli, yeast

Inserted sequence
DNA fragment
to be cloned
Vectors
The substance that can serve as carriers to allow
replication of recombinant DNAs.
 Plasmids
 Phage λ
 Plasmid phage hybrids
MCS
Plasmids
ORI
Ab. Resis
ds circles of DNA that can replicate autonomously.
Three features of the plasmid cloning vectors:
 Multiple cloning site. The place where foreign DNA
fragments can be inserted.
 An origin of replication. The replication origin is a specific
DNA sequence of 50-100 base pairs that must be
present in a plasmid for it to replicate. Host-cell enzymes
bind to ORI, initiating replication of the circular DNA.
 A gene specifying resistance to an Antibiotic. This
permits selective growth of the host cell.
Most often used: Resistance to ampicillin, penicillin,
tetracycline, kanamycin, and chloramphenicol.
Phage λ



A phage λ virion has a head, which
contains the viral DNA genome, and a
tail, which functions in infecting E.coli
host cells.
Advantages over plasmids: They infects
cells much more efficiently than
plasmids transform cells. The yield of
clones with vectors usually higher.
Because of its efficiency, phage λ is
often used in library construction.
Viral
Genome
The Cell
E.coli:


Normal E. coli cells cannot take up plasmid DNA from
the medium. Exposure to high concentration of certain
divalent cations, CaCl2, makes a small fraction of cells
permeable to foreign DNA.
Each component cell incorporates a single plasmid DNA
molecule, which carries an antibiotic-resistance gene.
When the cells are treated wit antibiotics on plates, only
a few of the transformed cells containing the antibioticsresistance gene on the plasmid vector will survive.
Inserted Sequence


Source of Nucleic acid to be cloned:
-DNA directly from organism
-DNA synthesized or amplified in vitro (cDNA or PCR
reactions).
-Previously cloned DNA. Generally a specific sequence.
Quality of DNA can be crucial
-Its purity, being free of contaminants
-its size is crucial when cloning very large pieces
Two Important Enzymes

Restriction Enzymes:
cuts the DNA from any organism at specific sequences
of a few nucleotides, generating a reproducible set of
fragments.

DNA Ligases:
insert DNA restriction fragments into replicating DNA
molecules producing recombinant DNA.
Mechanism
Restriction Enzyme: EcoRI
5`
G AAT T C
3`
5`
G
AAT T C
3`
C T TAA G
5`
3`
C T TAA
Cleavage
B 5`
OH
T T A A
3`
C 5`
3`
OH
T T A A
OH
C G
5`
OH
5`
P
+
5`
A
3`
G
Sticky ends
DNA Ligases
5`
3`
3`
P
3`
A A T T
P
3`
T T A A
P
5`
OH
Complementary
ends base-pair
DNA
ligases
P
OH
T A C G
P
5`
A A T T
3`
3`
T T A A
5`
+
Unpaired
B and C
Outline
 What is cloning?
 How to do cloning?
 Validation
 Application
Validation
Because introducing DNA into an organism is
usually not very efficient, we need to do
validation.
 Selection- A technique that isolates only a
particular type of cell or organism.
 Screen- A technique that allows identifying a
particular type of cell or organism but does not
isolate it from other types.
Selection
pBR322
Amp. R
EcoRI
Tet. R
Tet. R
Amp. R
Cell
Ampicillin
Screen
Cell
pBR322
EcoRI
Amp R.
EcoRI
Tet. R
EcoRI
Tet. R
Add Amp.
Tetracycline
Replica
plating
process
The cells we want
Other validation methods
Promoter
Ori.
About 100 bp
Multi. cloning site
EcoRI
U.P.
BamHI
SalI
EcoRI
+
Ab. Resis.
EcoRI
Inserted Gene
About 500bp-5kb
Marker
L.P.
PCR
With Prod. Without Prod.
Restriction
Enzyme
Marker
With Prod. Without Prod.
Sequencing
Dideoxy: (Sanger) Manual






Primer extension reactions in four separate tubes.
Using a dideoxy nucleotide as the chain terminator.
Each tube contains different dideoxy nucleotide (ddATP,
ddCTP, ddGTP, ddTTP).
Radioactive dATP is also included in all the tubes so the
DNA products will be radioactive.
The results is a series of fragments of different lengths.
Finally, autoradiography is performed to visualize the
DNA fragments.
Dideoxy: (Sanger) Manual
a) Primer extension reaction
c) Electrophoresis of the Protein
ddA
TACTATGCCAGA
20-base primer
25-base primer
ddC
ddG
ddT
T
C
T
G
G
C
A
T
A
G
T
A
Replication with ddTTP
TACTATGCCAGA
ATGA T
b) Product of the four reactions
Product of ddA rxn
Template:
(21)
(24)
(26)
TACTATGCCAGA
A
ATGA
ATGATA
Product of ddG rxn
Template:
(23)
(28)
(29)
TACTATGCCAGA
ATG
ATGATACG
ATGATACGG
Product of ddC rxn
Template:
(27)
(31)
TACTATGCCAGA
ATGATAC
ATGATACGGTC
Product of ddA rxn
Template:
(22)
(25)
(30)
(32)
TACTATGCCAGA
AT
ATGAT
ATGATACGGT
ATGATACGGTCT
Sequencing
Dideoxy: (Sanger) automated




The “manual” sequencing technique is powerful but slow,
thus Rapid automated sequencing methods are required.
Still based on the procedure using dideoxy nucleotides,
but tagged with a different fluorescent molecule, so the
product from each tube will emit a different color
fluorescence when excited by light
After extension reaction and chain termination, all 4
solutions are mixed and electrophoresed together in the
same lane on gel analyzed by laser beam
The color of the fluorescent light emitted from each
oligonucleotide is detected electronically
Outline
 What is cloning?
 How to do cloning?
 Validation
 Application
Application
Expression
Library
Expression
Why?
You want the cloned gene to make its product, normally a
protein.
 Identifying gene from library requires expression.
 To overproduce the protein and purify it.
 For in vivo studies of the protein.
Expression
Expression Vectors:
Vectors that can yield the protein products of the cloned
genes.
Two elements that are required for active gene
expression: a strong promoter and a ribosome binding
site near an initiating ATG codon.
The main function of an expression vector is to yield the
product of a gene, therefore a strong promoter is
necessary. The more mRNA is produced, the more
protein product is made.
Inducible Expression Vectors



Protein produced in a large quantity in bacteria can be
toxic, so it is advantageous to keep a cloned gene
repressed before expressing it.
Solution: keep the cloned gene turned off by placing it
downstream of an inducible promoter that can be turned
off.
IPTG strongly induce lac promoter
Expression
Expression Vector that Produce Fusion Proteins
 Fusion proteins: Gene (or part of a gene) for one
protein fused to part or all of a gene for a second protein.
Major uses for generating fusion proteins:
 The ‘tag’ of the fusion protein can greatly aid biochemical
purification. If the tag binds a particular substance, a
column prepared containing that bound substance can
be used to purify the tagged protein from virtually all
other proteins.
(His)6
ATG
MCS
Oligohistidine regions like this have a
high affinity for metals like nickel, so
proteins that have such regions can be
purified using nickel affinity
chromatography
Fusion Protein


The ‘tag’ can serve as a convenient way for
identification of the tagged protein in cells or extracts.
For example, a short peptides can be sufficient to use as
a tag for antibody binding. In this way, a common
antibody can be used, eliminating the need to develop a
novel reagent specific to the protein of interest.
The ‘tag’ can be used as a surrogate in the
quantification of the tagged protein. Again, a routine
assay of the activity of the tag can be used to monitor
amounts of a protein of interest that may have no means
of assay otherwise.
Library Construction
A library is a collection of different cloned DNAs
from a single source that are present in different
copies of a particular cloning vector.


Genomic library – for genome sequencing
cDNA library – derived from mRNA of a
particular tissue, for isolating specific genes
Library Construction
The principle of library construction is basically quite simple.
 Cut a DNA vector at a unique restriction site and ligate
into it the DNA that you want to make a library out of.
 If you want a library of human genomic DNA, you use
fragmented human DNA.
 The ligation mix is not yet considered the library. The
library comes after the generation of E. coli cells carrying
the cloned DNA.
 To generate a library with a million clones for example,
you need to recover a million independent colonies
carrying plasmids or a million independent phage
plaques. By pooling together all the independent clones
you get the library.
Library Construction
Though simple in principle, libraries are difficult to make well.
 Partly this is just a matter of scale. While in routine
cloning, you generally just need to recover a single type of
clone, a library has to generate very large numbers of
independent DNA inserts.
 While used pretty frequently, libraries are seldom made.
Few people have much experience.
 The best advice for making a library is to not do it unless
you really have to. Get a library from someone else that
has already made one. Some are commercially available.
Thanks
Dr. Robert A. Scott
All the members in the group
Xiaoming Wang
Reference
Manuscript of Course Genetics 8920
 Molecular Biology
Robert F. Weaver
 Molecular Cell Biology
Lodish, Berk, Zipursky, Matsudaira,
Baltimore, Darnell
