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Cloning
Guanqun Yuan12-13-2004R.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 identi
cal twin is not a clone. DNA sequence amplified by growth is a clone, I
dentical DNA molecules produced in vitro (a PCR rxn) is not a clone.
Outline
What is cloning? How to do cloning? Validation Application
Plasmid vector
+
DNA fragment to be cloned
Enzymatically insert DNA into plasmid vector
Recombinant plasmid
Mix E.coli cells with plasmids in presence of CaCl2 Culture on nutrient agar plat
es containing ampicillin
Cells that do not take up plasmids die on ampici
llin plates
Transformed E.coli cell survives
Bacterial chromosome
Independent plasmid
replication
Cell multiplication
How?Amp R.
Amp R.
ORI
ORI
Composition
Vectors Plasmids, or phage
The cell E.coli, yeast
Inserted sequence
Plasmid vector
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
Plasmids
ds circles of DNA that can replicate autonomously.Three features of the plasmid cloning vectors: Multiple cloning site. The place where foreign DNA fragm
ents 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.
MCS
Ab. ResisORI
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 th
e 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 antibiotics-resistance 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 react
ions). -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 o
f a few nucleotides, generating a reproducible set of fragments.
DNA Ligases: insert DNA restriction fragments into replicating DNA mol
ecules producing recombinant DNA.
5` G A A T T C 3`
Restriction Enzyme: EcoRI
3` C T T A A G 5`
Cleavage
5` G A A T T C 3`
3` C T T A A G 5`
Sticky endsDNA Ligases
3` T T A A P5` OH
+
3` T T A A P5` OH
3` C G P5` OH
3` T A C G P5` OH
A
C
B
Complementary ends base-pair
5` A A T T 3`
3` T T A A 5`
OH
P P
OH
+Unpaired B and C
5` A A T T 3`
3` T T A A 5`
DNA ligases
Mechanism
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
EcoRI
pBR322
Ampicillin
Tet. R
Amp. R
Tet. RAmp. R
Cell
Screen
EcoRIAmp R.
Tet. R
pBR322
EcoRITet. R
TetracyclineEcoRI
Replica plating process
Add Amp.
The cells we want
Cell
Other validation methods
Inserted Gene
EcoRIEcoRI
Promoter
Ori.
Multi. cloning site
Ab. Resis.
EcoRI
About 100 bp
About 500bp-5kb
BamHI
SalIPCR
U.P.
L.P.
Marker 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 DN
A fragments.
Dideoxy: (Sanger) Manuala) Primer extension reaction
TACTATGCCAGA
20-base primer Replication with ddTTP
TACTATGCCAGA
25-base primerATGAT
b) Product of the four reactions
TACTATGCCAGAA
Template:(21)(24) ATGA
ATGATA(26)
Product of ddA rxn
TACTATGCCAGAATG
Template:(23)(28) ATGATACG
ATGATACGG(29)
Product of ddG rxn
TACTATGCCAGAATGATAC
Template:(27)(31) ATGATACGGTC
Product of ddC rxn
TACTATGCCAGAAT
Template:(22)
(32)
ATGATATGATACGGT(30)
Product of ddA rxn
(25)
ATGATACGGTCT
c) Electrophoresis of the Protein ddA ddC ddG ddT
TCTGGCATAGTA
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
ExpressionLibrary
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 g
enes.Two elements that are required for active gene expressio
n: 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
ExpressionExpression 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.
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
ATG
(His)6
MCS
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 particula
r 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 int
o it the DNA that you want to make a library out of. If you want a library of human genomic DNA, you use fra
gmented human DNA. The ligation mix is not yet considered the library. The libra
ry 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 8920Molecular Biology
Robert F. WeaverMolecular Cell Biology
Lodish, Berk, Zipursky, Matsudaira,
Baltimore, Darnell