Download Molecular Cloning

Lecture 13
Molecular Cloning
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Recombinant DNA technology depends on the ability to produce
large numbers of identical DNA molecules (clones).
Clones are typically generated by placing a DNA fragment of
interest into a vector DNA molecule, which can replicate in a
host cell.
When a single vector containing a single DNA fragment is
introduced into a host cell, large numbers of this fragment are
reproduced along with the vector.
Two commonly used vectors for cloning are E. coli plasmid
vectors and bacteriophage λ vectors
DNA restriction cutting, plasmid, phage, DNA ligation,
antibiotic selection, cDNA, cDNA Library, expression vector
Ligase reaction: opposite to restriction enzyme, requires 5’-P (from DNA2) and 3’-OH (from DNA1).
Ligase mechanism:
DNA ligase forms activated
ligase-AMP (from ATP or NAD),
AMP links to 5’-P, 3’-OH
attacks forming new
phosphodiester bond, seal!
Ligation of the sticky ends:
two DNA molecules, cleaved with
EcoRI and ligate to form
recombinant molecules
Formation of cohesive ends
Addition and cleavage of a chemically synthesized linker.
Producing sticky ends with adaptors.
Plasmid and cloning vector
Plasmid: circular, ds, extrachromosomal self-replicating DNA molecule occuring naturally in
bacteria, yeast and higher eukaryotic cells, either parasitic or symbiotic, ~kb to 100 kb, at least
one to the daughter cell (drug-resistance, transfer genes encoding proteins forming
macromolecular tube or pilus).
Cloning vector: engineered plasmids with reduced size (~3 kb), containing only ori, drugresistance gene, cloning site. Ampr encodes β-lactamase which inactivates ampicillin.
Basic cloning
cut both of the vector and insert with BamH1,
vector treated with alkaline phosphatase
(prevent resealing),
ligase put insert into the vector, two nicks will be
sealed in vivo.
Recombinant DNA
The plasmid and the
foreign DNA are cut by a
restriction endonuclease
(EcoRI in this example)
producing intermediates
with sticky and
complementary ends.
Those two intermediates
recombine by basepairing and are linked by
the action of DNA ligase.
A new plasmid containing
the foreign DNA as an
insert is obtained. A few
mismatches occur,
producing an undesirable
recombinant.
1973 PNAS 70, 3240
First recombinant DNA produced:
Stanley Cohen and Herbert Boyer cut 2 plasmids with
EcoRI, ligate and screen for E.coli clones that are
resistant to both antibiotics since they harbor the
recombinant plasmids. Patent 4,237,224, 1980
http://www.dnai.org/text/mediashowcase/index2.html?id=1186
pBR322 plasmid
Host containing pBR322 with insert at different restriction sites can be selected.
There are many sites can be used.
Cloning at the PstI site
Make construct, transform
bacteria, screen for
tetracycline-resistant but
ampicillin-sensitive clones
using replica plating (make a
replica plate treating with
ampicillin, sensitive clones
are recovered from the
original plate).
Polylinkers
Chemically synthesized polylinker
containing one copy of several
different restriction sites is
introduced into the vector to
facilitate directional cloning.
MCS: multiple cloning site
MCS is inserted into a gene encoding the N-terminal part of β-galactosidase.
Clones harboring the vector plus an insert remain white.
pUC series: derived from pBR322 (40% deleted), the MCS is in the lacZ’ (Nterminal of β-galactosidase, the host contains the C-terminus portion, MCS starts
after the 7th codon, retains the ORF), so no insert blue (with X-gal), with insert
white.
Molecular cloning
The general procedure for cloning with plasmid vectors:
plasmid with Ampr and insert is transformed into host with
CaCl2 or other methods, grow in ampicillin plate, surviving
cells form colony.
Plasmid cloning permits isolation of DNA fragments from
complex mixtures: Each colony is derived from a single
cell containing the same plasmid.
Cloning strategy
Process by which a plasmid is used to import recombinant DNA into a host cell for cloning.
In DNA cloning, a DNA fragment that contains a gene of interest is inserted into a cloning
vector or plasmid. The plasmid carrying genes for antibiotic resistance, and a DNA strand,
which contains the gene of interest, are both cut with the same restriction endonuclease.
The plasmid is opened up and the gene is freed from its parent DNA strand. They have
complementary "sticky ends." The opened plasmid and the freed gene are mixed with
DNA ligase, which reforms the two pieces as recombinant DNA.
This recombinant DNA stew is allowed to transform a bacterial culture, which is then
exposed to antibiotics. All the cells except those which have been encoded by the plasmid
DNA recombinant are killed, leaving a cell culture containing the desired recombinant
DNA.
DNA cloning allows a copy of any specific part of a DNA (or RNA) sequence to be
selected among many others and produced in an unlimited amount. This technique is the
first stage of most of the genetic engineering experiments: production of DNA libraries,
PCR, DNA sequencing, et al.
Molecular cloning
Molecular cloning
DNA Libraries in λ phage and other
cloning vectors
• Cloning all of the genomic DNA of higher organisms
into plasmid vectors is not practical due to the
relatively low transformation efficiency of E. coli and
the small number of transformed colonies that can be
grown on a typical culture plate
• Cloning vectors derived from bacteriophage do not
suffer from such limitations
• A collection of clones that includes all the DNA
sequences of a given species is called a genomic
library
• A genomic library can be screened for clones
containing a sequence of interest
(i) EM of bacteriophage λ virion.
(ii) Simplified view of bacteriophage genome:about 60 genes, replaceable region
not essential and can be replaced by foreign DNA up to 25 kb. Large segment of
the 48kb DNA of the λ phage are not essential for productive infection and can be
replaced with inserts.
(iii)Assembly of bacteriophage λ virion: during late stage of λ infection
concatomers are formed, Nu 1 and A protein push 1 copy of λ from concatomer
into the head, then tail added and they are ready to go to the war.
cloning
λ phage vector
Charon phage 4 takes 20 kb,
remove the stuffer,
ligate the insert,
mix with the in vitro
packaging extract,
infect cells,
the insert has to be between
12 kb and 20 kb.
Alternative infection modes for λ phage:
lytic or lysogenic pathway
A set of lambda (or plasmid)
clones that collectively
contain every DNA
sequence in the genome of
a particular organism.
Nearly complete genomic
libraries of higher
organisms can be prepared
by lambda cloning.
Genomic Library
(i) A set of lambda (or plasmid) clones that
collectively contain every DNA sequence in
the genome of a particular organism. Nearly
complete genomic libraries of higher
organisms can be prepared by lambda
cloning.
(ii) Construction of a genomic library:
Sau3A and BamH1 generate
complementary sticky ends.
(iii) Plaque hybridization: selection of positive
clones, filter replicates the dish, plaque containing
phage DNA denatured by base, block the filter with nonspecific DNA or protein, hybridize with specific gene
probe, autoradiography to select the target .gene.
(iv) The most common approach to identifying a
specific clone involves screening a library by
hybridization with radioactively labeled DNA or
RNA probes. Identification of a specific clone
from a l phage library by membrane
hybridization. Identifying, analyzing, and
sequencing cloned DNA
Cosmid vector
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Larger DNA fragments up to 45
kb can be cloned in cosmids and
other vectors,
cosmid has cohesive ends for
packaging and plasmid ori for
replication as plasmids in
bacteria.
cosmids cannot replicate as
phages but they are still
infectious.
M13 Phage
M13 Phage: a filamentous
virus 900 nm long and 9 nm
wide, single strand 6.4kb
circle DNA protected by 2710
identical proteins, gets into E.
coli via sex pilus, replicate to
RF, only (+) is packed into
virus particle. Useful for
sequencing.
Complementary DNA
DNA (cDNA) libraries
are prepared from isolated
mRNAs
Complementary
oligo-dT column to purify mRNA
Complementary DNA (cDNA) libraries
Formation of cDNA duplex: RT, alkali digestion, oligo(dG) tailing, oligo(dC) primer
Preparation of a cDNA library
Making a cDNA Library
Making a cDNA Library: Reverse
Transcriptase, RNase H (degrade RNA in
RNA-DNA hybrid) , DNA polymerase I (using
RNA as primer and perform nick translation),
Terminal deoxynucleotidyl transferase (adding
dCTP to the cDNA duplex, and dGTP to the
vector), Ligase and pol I in the host perform
ligation, RNA removal, and sealing.
Use RT-PCR to clone a
single cDNA if the sequence
of mRNA is known.
Phagemid
pBS or pBluescript, MCS inserted into lacZ’, ori of the ss phage f1, T3 and T7 phage
RNA polymerase promoters.
Expression vector
The expression vector contains a fragment of the E. coli chromosome containing the
lac promoter and the neighboring lacZ gene. In the presence of the lactose analog
IPTG, RNA polnormally transcribes the lacZ gene, producing lacZ mRNA, which is
translated to the encoded protein, β-galactosidase.
(i) Forming fusion protein in plasmid vector: pUC and pBS vectors place inserted
DNA under the control of the lac promoter. If the DNA is in frame with the lacZ’
gene, a fusion protein will be expressed.
E. coli expression systems can
produce full-length proteins:
Producing high levels of proteins
from cloned cDNAs. Many
proteins are normally expressed at
very low concentrations within
cells, which makes isolation of
sufficient amounts for analysis
difficult. To overcome this
problem, DNA expression vectors
can be used to produce large
amounts of full length proteins.
Lac promoter.
The lacZ gene can be cut out of the expression vector with restriction enzymes
and replaced by the G-CSF cDNA. After transformation, IPTG will induce the
expression of G-CSF protein.
Two-step system : T7 RNA polymerase and T7 late promoter
Even larger amounts of a desired protein can be expressed with a two-step
system, 10-70% of the total protein synthesized by these cells after IPTG is the
protein of interest.
Forming fusion protein in phage vector: λg11with lac control region followed by lacZ gene,
cloning sites are located within the lacZ gene, direct detection of protein with antibody.
Degenerate probe: screen for the correct cDNA or genomic
clone from the library.