2. Recombinant DNA Technology- Recap
• A set of techniques for recombining genes
from different sources in vitro and
transferring the recombinant DNA into cells
where it may be expressed.
• Use of recombinant DNA techniques allows
modern biotechnology to be more precise and
systematic than earlier research results.
3. Recombinant DNA
• It allows genes to be moved across species
barriers – hence a powerful tool
• Our understanding of eukaryotic molecular
biology has been enhanced
• It has been applied in the Human Genome
Project – to transcribe and translate the entire
human genome in order to understand the
human organism
• The ultimate goal is the improvement of human
health
4. Gene (DNA) Cloning
• Recombinant DNA technology makes it possible to clone
genes for basic research and commercial applications
• Recombinant DNA technology allows scientists to examine
the structure and function of the eukaryotic genome
because it contains:
Biochemical tools for the construction of recombinant DNA
Methods for purifying DNA molecules and proteins of interest
Vectors for carrying recombinant DNA into cells for replication
Techniques for determination of nucleotide sequences of DNA
molecules
6. Restriction enzymes are used to make
Recombinant DNA
• Restriction enzymes were first discovered in the 1960s
• They occur naturally in bacteria where they protect the
bacterium against foreign invading DNA from other
organisms (e.g. Viruses or phages)
• The foreign DNA is restricted by it being cut into small
segments – thus restriction is the process of cutting of
foreign DNA into small pieces.
• Most restriction enzymes only recognise small short
sequences (4-8 nt) called recognition sites and only cut
at specific points within these sequences
8. Restriction enzymes
• Recognition sequences are symmetric in that the same sequence of
nucleotides is found on both strands, but run in opposite directions
• They usually cut phosphodiester bonds of both strands in a staggered
manner, so that the resulting dsDNA fragments have single stranded
ends, referred to as sticky ends
• The sticky ends form hydrogen-bonded base pairs with complementary
sticky ends on other DNA molecules
• These unions are temporary since they are only held by a few hydrogen
bonds
• The bonding is made permanent by DNA ligase, which catalyses the
formation of covalent phosphodiester bonds
• This result is the same as natural genetic recombination, the production of
recombinant DNA – a DNA molecule carrying a new combination of
genes
9. Cloning Vectors
• A Cloning Vector is a DNA molecule that can
carry foreign DNA into a cell and replicate in
the cell
• The two most often used vectors are
bacterial plasmids and viruses (phages)
10. Cloning with Plasmid Vectors
• Plasmids are circular, dsDNA molecules that
separate from a cell’s chromosomal DNA.
• They occur naturally in bacteria
• Plasmids exist in parasitic or symbiotic
relationship with their host cells. Their sizes
range from a few thousand bp to more that
100kb.
• They duplicate before every cell division.
• During cell division, at least, one is segregated
into each daughter cell.
11. Cloning with Plasmid Vectors
• Many plasmids also contain transfer genes coding for
proteins that transfer a copy of the plasmid to other
host cells of the same or related bacteria species, by
conjugation (E. coli plasmids can be engineered for use
as cloning vectors)
• Plasmid most commonly used in recombinant DNA
technology replicate in E. coli. These plasmids have
been engineered to optimize their use as vectors in
DNA cloning
• Their length (circumference) is reduced to range form
1.2 – 3 kb - much less than that occurring naturally in E
coli plasmids
12. Cloning with Plasmid Vectors
• Most plasmid vectors contain little more than the essential
nucleotide sequences required for their use in DNA cloning.
• They contain a replication origin (ORI), a drug-resistance
gene, and a region in which exogenous DNA fragments
can be inserted
• The ORI is a specific DNA sequence of 50-100 bp that must
be present in a plasmid for it to replicate.
• Host enzymes bind to ORI, initiating replication of the
circular plasmid
• Thus, any DNA sequence inserted into such a plasmid is
replicated along with the rest of the plasmid DNA, the basis
of molecular DNA cloning.
13. Cloning with Plasmid Vectors
• Steps of recombinant plasmid formation
Production of restriction fragments
Ligation of restriction fragments to plasmid DNA
Transformation of antibiotic-sensitive E. coli cells
with recombinant plasmids.
• Other cloning vectors are bacterial artificial
chromosomes (BACs) and yeast artificial
chromosomes (YACs)
15. Plasmid Cloning
• Remember the Genetic code is the same in all
Organisms
• Plasmids commonly used in recombinant DNA
technology replicate in E.coli
• These plasmids have been engineered to
optimise their use as vectors for DNA cloning
• Their length range from 1.2 to 3.0 kb
• They contain a replication origin (ORI), a drug
resistance gene and a region for insertion of
exogenous DNA
16. Transformation of the host cell
• Transformation is genetic alteration of a cell
caused by the uptake and expression of
foreign DNA regardless of the mechanism
involved
• Transformation permits plasmid vectors to be
introduced into and expressed in E. coli cells
• A plasmid vector must contain a drug-
resistance gene coding for an enzyme that
inactivates a specific antibiotic in order to be
useful in DNA cloning.
17. Transformation of the host cell
• The ability to select transformed cells is
critical to DNA cloning because the
transformation of E.coli with isolated plasmid
DNA is inefficient
• The cells must be exposed divalent cations
such as Ca2+
to make the cells permeable
• E.coli are treated with CaCl2 and mixed with
plasmid vecotrs
• Frequently, only 1 cell out of 10 000 or more
cells beomes competent to take up foreign
DNA
18. Transformation of the host cell
• Each cell takes up a single recombinant plasmid DNA
molecule
• The treated cells are plated on a Petri dish of
nutrient agar containing the antibiotic
• Only the transformed cells containing the antibiotic –
resistance gene on the plasmid vector will survive
• Thus all the plasmids in such a colony of selected
transformed cells are descended from a single
plasmid taken up by cells that form the colony
19. Isolation of DNA Fragment From a Mixture
• The initial fragment of DNA inserted into the
parental plasmid is referred to as cloned DNA since it
can be isolated from the cloned cells
• DNA cloning allows a particular nucleotide sequence
to be isolated from a complex mixture of fragments
with many different sequences.
• For example, assume we have 4 different types of
DNA fragments each with a unique sequence.
• Each fragment type is inserted alone into a plasmids
vector.
20. Isolation of DNA Fragment From a Mixture
• The resulting mixture of recombinant plasmids is
incubated with E. coli cells treated with CaCl2.
• The cells are then cultured on antibiotic selective
plates. (e.g. plates containing ampicillin).
• Each colony that develops arises from a single cell
that took up one or the other recombinant plasmids.
• All the cells in a given colony thus carry the same
DNA fragment.
21. Isolation of DNA Fragment From a Mixture
• Overnight incubation of E. coli at 37°C produces
visible colonies.
• These colonies are isolated from each other on the
culture plate. Hence, copies of the DNA fragments
in the original mixture are separated in the
individual colonies.
• Therefore, DNA cloning is a powerful but simple
method for purifying a particular DNA fragment
from a complex mixture of fragments and
producing large numbers of the fragment of
interest.
• Each transformed cell contains multiple copies of a
22. Production of recombinant Plasmids
• To clone specific DNA fragments in a plasmid (or any
other) vector, the fragments must be produced and
then inserted into the DNA vector.
• Restriction enzymes and DNA ligases are utilized to
produce such recombinant cloning vector.
• Restriction enzymes are bacterial enzymes that
recognize specific 4 to 8 bp sequences (restriction
sites), and then cleave both DNA strands at this site.
• These enzymes cut within the DNA molecule hence
they are called endonucleases, to distinguish them
from exonucleases, which digest nucleic acids from
an end.
23. Production of recombinant Plasmids
• Many restriction sites are short inverted repeat
sequences. That is, the restriction site sequence is
the same on each DNA strand when read in the 5′
→3 direction.′
• The DNA digest (fragments) produced by restriction
enzymes are called restriction fragments.
• Modification enzymes protect bacterial DNA from
cleavage by restriction enzymes, by adding a methyl
group to one or two bases usually within the
restriction site.
24. Production of recombinant Plasmids
• Many restriction enzymes generate fragments
that have a single stranded tail at both ends.
• These tails are complementary to those on all
other fragments generated by the same
restriction enzyme.
• These tails are referred to as sticky ends (also
called cohesive ends) and can transiently base
pair at room temperature with those on other
DNA fragments generated with the same
restriction enzyme, regardless of the source of
the DNA molecules.
25. Production of recombinant Plasmids
• The base paring of sticky ends permits DNA from
widely different species to be ligated, forming
chimeric molecules
• Purified DNA ligase is used to covalently join the
ends of restriction fragments in vitro.
• DNA ligase can catalyze the formation of a 5 →3′ ′
phosphodiester bond between 3 - OH end of one′
restriction fragment strand and the 5 - PO′ 4 end of
another restriction fragment strand during the time
that the sticky ends are transiently base-paired.
26. Production of recombinant Plasmids
• Plasmids vectors containing a polylinker (or
multiple cloning site sequence) are commonly
used to produce recombinant plasmids
carrying exogenous DNA fragments.
• Polylinkers are chemically synthesized and
then introduced into the plasmid vector.
27. Production of recombinant Plasmids
• Since the polylinker contains several different
restriction sites, one of the restriction
enzymes whose recognition sites is in the
polylinker is used to cut both the plasmid
molecules and genomic DNA.
• This generates singly cut plasmids and
restriction fragments with complementary
sticky ends.
28. Production of recombinant Plasmids
• In the presence of DNA ligase, DNA fragments
produced with the same restriction enzyme
will be inserted into the plasmid.
• The ratio of DNA fragments to be inserted to
cut vectors and other reaction conditions are
chosen to maximize the insertion of one
restriction fragment per plasmid vector.
29. Production of recombinant Plasmids
• The recombinant plasmids produced in in vitro
ligation reactions are then used to transform
antibiotic sensitive E. coli cells.
• All cells in each antibiotic-resistant clone that
remains after selection contain plasmids with
the same inserted DNA fragment, but
different clones carry different fragments.
30. Steps of transformation
• Production of restriction fragments
• Ligation of restriction fragments to plasmid
DNA
• Transformation of antibiotic-sensitive E. coli
cells with recombinant plasmids
33. Cloning with Bacteriophage)
λ-Phage
• Most cloning done with E. coli plasmid
because of the relative simplicity of the
procedure
• However, the number of individual clones that
can be obtained by this method is limited by:
(1)the low efficiency of E. coli transformation
and (2) the small number of individual
colonies that be detected on a Petri dish.
34. Cloning with Bacteriophage)
λ-Phage
• The limitations make plasmid cloning of all
genomic DNA of higher organisms impractical
• For example, 1.5 x105
clones carry 25 kb DNA
fragments are required to represent the total
human genome
• Cloning vectors from bacteriopage λ have
proved to be a more practical means for obtaining
the required number of clones to represent large
genomes
35. Cloning with Bacteriophage)
λ-Phage
• Such a collection of λ clones that includes all
the DNA sequences of a given species is called
a genomic library.
• A genomic library can be screened for λ
clones containing a sequence of interest.
• Bacteriophage λ can be modified for use as a
cloning vector and assembled in vitro.
36. Cloning with Bacteriophage)
λ-Phage
• A λ-phage virion has a head region, which
contains the viral DNA.
• It also has a tail region, which enables the λ-
phage to infect E. coli host cells.
• Only the λ DNA enters the cell when a λ virion
infects a host cell.
• The viral DNA then undergoes either lytic or
lysogenic growth.
37. Cloning with Bacteriophage)
λ-Phage
• In lytic growth, the viral DNA is replicated and
assembled into more than 100 progeny virions
in each infected cell.
• This kills the cell in the process and releases
the replicated virions.
• In lysogenic growth, the viral DNA inserts in
the bacterial chromosome where it is
passively replicated along with the host-cell
chromosome as the cell grows and divides.
38. Cloning with Bacteriophage)
λ-Phage
• In the Lysogenic Cycle:
Viral DNA merges with Cell DNA and
does not destroy the cell.
The Virus does not produce progeny.
There are no symptoms of viral
infection.
Temperate viral replication takes
place.
39. Cloning with Bacteriophage)
λ-Phage
• In the Lytic Cycle:
Viral DNA destroys Cell DNA, takes over cell
functions and destroys the cell.
The Virus replicates and produces progeny
phages.
There are symptoms of viral infection.
Virulent viral infection takes place.
41. Cloning with Bacteriophage)
λ-Phage
• The λ genes coding for the head and tail
proteins as well as various proteins involved in
the lytic and lysogenic growth pathways are
clustered in discrete regions of about 50kb
viral genome.
• The genes in the lysogenic pathway are not
relevant for use of bacteriophage λ as a
vector.
42. Cloning with Bacteriophage)
λ-Phage
The λ proteins, designated Nu1 and A, bind to
COS sites and direct insertion of the DNA
between 2 adjacent COS sites into a
preassembled head.
43. Cloning with Bacteriophage)
λ-Phage
• Genes in the lysogenic pathway are not
relevant for use of bacteriophage λ as a
vector.
• Hence, they are removed from the viral DNA
and replaced with other DNA sequences of
interest.
• Up to 25 kb of foreign DNA then can be inserted
into λ genome, resulting in a recombinant DNA that
can be packaged to form virions capable of
replicating and forming plaques in E. coli host cells.
44. Cloning with Bacteriophage)
λ-Phage
• The key to the high efficiency of λ-phage cloning is
the ability to assemble λ virions in vitro.
• Viral heads and tails initially are assembled
separately from multiple copies of the various
proteins that compose these complex structures.
• λ DNA replication in the host cell generates
concatemers - multimeric DNA molecules that
consist of multiple copies of the viral genome linked
end-to-end and separated at COS sites.
45. COS sites are protein-binding
nucleotide sequences that occur once
in each copy of the λ genome.
46. Cloning with Bacteriophage)
λ-Phage
• The host-cell chromosomal DNA is not
inserted into the λ heads because it does not
contain copies of the COS sequence.
• Only one λ DNA is inserted into a
preassembled λ head.
• After insertion of the λ DNA the preassembled
tail is attached producing complete virions.
47. • Preparation of recombinant infectious λ
virions:
The phage-assembly process is carried out in
vitro.
E. coli cells are infected with a mutant defective
in A protein (one of the 2 proteins required for
packaging λ DNA into preassembled phage heads).
These cells then accumulate Empty heads
Preassembled tails also accumulate since they
only attach to heads filled with DNA
48. Cloning with Bacteriophage)
λ-Phage
• The cells are lysed experimentally and an
extract containing high concentrations of
heads and tails is prepared.
• When this extract is mixed with A protein and
recombinant λ DNA containing a COS site, the
DNA is packaged into the empty heads.
• The tails in the extract then combine with the
filled heads, yielding complete virions carrying
the recombinant λ DNA.
49. Cloning with Bacteriophage)
λ-Phage
• The recombinant virions produced by this
method are fully infectious and can efficiently
infect E. coli cells.
• Each virion particle binds to receptor on the
surface of a host cell and injects it packed
recombinant DNA into the cell.
• This infection process is about 1000x more
efficient than transformation with plasmid
vector.
.
50. Cloning with Bacteriophage)
λ-Phage
• ≈106
colonies per µg in plasmid transformation
compared to ≈ 109
plaques representing λ-
phage representing λ clones for λ-phage
transformation per µg of recombinant DNA
• a collection of λ clones that includes all the
DNA sequences of a given species is called a
genomic library. A genomic library can be
screened for λ clones containing a sequence
of interest.
51.
52.
53.
54. Identification of a specific clone from a λ phage library
by membrane hybridization to a radiolabeled probe
• The position of the signal on the autoradiogram
identifies the desired plaque on the plate.
• In practice, in the initial plating of a library the
plaques are not allowed to develop to a visible size so
that up to 50,000 recombinants can be analyzed on a
single plate.
• Phage particles from the identified region of the plate
are isolated and replated at low density so that the
plaques are well separated.
• Then pure isolates can be obtained by repeating the
plaque hybridization as shown in the figure.
