 DNA cloning is a technique for reproducing

DNA fragments.
 It can be achieved by two different
approaches:
▪ cell based
▪ using polymerase chain reaction (PCR).
 a vector is required to carry the DNA
fragment of interest into the host cell.
 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
 Massive amplification of DNA sequences
 Stable propagation of DNA sequences
 A single DNA molecule can be amplified
allowing it to be:
▪ Studied - Sequenced
▪ Manipulated - Mutagenised or Engineered
▪ Expressed - Generation of Protein
 Gene of interest is
cut out with RE
 Host plasmid is
cut with same RE
 Gene is inserted
into plasmid and
ligated with ligase
 New plasmid
inserted into
bacterium
(transform)
 Separating DNA from other cellular components
such as proteins, lipids, RNA, etc.
 Avoiding fragmentation of the long DNA molecules
by mechanical shearing or the action of endogenous
nucleases
 Effectively inactivating endogenous nucleases
(DNase enzymes) and preventing them from
digesting the genomic DNA is a key early step in
the purification process. DNases can usually be
inactivated by use of heat or chelating agents.
 Key Steps
 Lysis of the cells
 Removal of contaminants
includes
 Proteins
 RNA
 Other
macromolecules
 Concentration of purified
DNA
1. Lysis of the cell

Use Detergent to solubilize the membrane lipid.

 DNA must be separated from proteins and cellular
debris.
Separation Methods
a) Organic extraction
b) Salting out
Traditionally, phenol: chloroform is used to extract DNA.
When phenol is mixed with the cell lysate, two phases form.
DNA partitions to the (upper) aqueous phase, denatured
proteins partition to the (lower) organic phase.
 Phenol: Denatures proteins and solubilizes denatured
proteins
 At high salt concentration, proteins are dehydrated,
lose solubility and precipitate.
Usually sodium chloride, potassium acetate or
ammonium acetate are used.
 Precipitated proteins are removed by centrifugation
 DNA remains in the supernatant.
Salting out method:
Cell lysis.
Protein digestion by proteinase enzyme.
Protein precipitation by high salt concentration.
Centrifugation will remove the precipitated proteins.
The supernatant contains the DNA.
DNA is then precipitated by adding ethanol.
The precipitated DNA is resuspended in the desired
buffer.
 Ethanol precipitation:
-Precipitation of DNA: Absolute Ethanol is layered on the top of
concentrated solution of DNA
- Fibers of DNA can be withdrawn with a glass rod
- Washing of DNA
- Desalt DNA: Most salts are soluble in 70% ethanol
 2- Use of Commercial DNA purification kits:

 The common lysis solutions contain

A. sodium chloride
B. Trimethamine (also known as tris ) , which is a
buffer to retain constant pH
C. Ethylendiaminetetraacetic (EDTA) , which binds
metal ions
D. Sodium dodecyl sulfate (SDS) which is a detergent .
E. An enzyme used in DNA extraction is protienase K
 Magnetic beads are coated with DNA antibodies or silica
to bind to DNA.
 Samples are lyses & and then treated with proteinase K.
 The lysates are then applied to the beads.
 Resin is subsequently washed & DNA is eluted of it at 65c
 Magnetic beads are separated from the sample on a
magnetic stand.
 1- Cell lysis , to expose the DNA within .
2- removing membrane lipids by adding a detergents or
surfactants .
3- removing proteins by adding a protease .
4- removing RNA by adding an Rnase.
5- precipitating the DNA with alcohol- usually ice cold
ethanol. In these alcohols , DNA strand will aggregate
together, giving a pellet upon centrifugation . This step also
removes alcohol- soluble salt.
 DNA concentration can be determined by measuring the
intensity of absorbance with a spectrophotometers &
comparing to a standard curve of known DNA
concentration.
 Measuring the intensity of absorbance of the DNA
solution at wavelength 260nm & 280nm is used as a
measure of DNA purity
 DNA purity: A260/A280 ratio: 1.7 – 1.9
 DNA concentration (μg/ml): A260 X 50
 DNA yield:
DNA conc. X Total volume of DNA solution
• DNA concentrations can be measured by ultraviolet
absorbance spectrophotometry

• Absorbance is measured at 260 nm.

A260 of 1 corresponds to 50 µg of double stranded DNA per

ml.

• With a pure sample of DNA, A260/A280 is 1.8

• Ratios of less than 1.8 indicates that the preparation is

contaminated with protein or with phenol
 Checking for Degradation DNA
 Running your sample through an agarose gel is a
common method for examining the extent of DNA
degradation. Good quality DNA should migrate as a
high molecular weight band, with little or no evidence
of smearing.
 DNA absorbs UV light at 260 &280 nm & aromatic
proteins absorb UV light at 280 nm Apure sample of
DNA has the 260/280 ratio at 1.8 & is relatively free
from protein contamination.
 A DNA preparation that is contaminated with
protein will have a 260/280 ratio lower than 1.8
Hypochromism. (A) Single-stranded DNA absorbs light more effectively than
does double-helical DNA. (B) The absorbance of a DNA solution at a
wavelength of 260 nm increases when the double helix is melted into single
strands.
DNA Probes and Genes Can Be Synthesized
by Automated Solid-Phase Methods
DNA strands, like polypeptides, can be synthesized by the sequential addition of
activated monomers to a growing chain that is linked to an insoluble support.

The activated monomers are protonated deoxyribonucleoside 3 - phosphoramidites.

In step 1, the 3 phosphorus atom of this incoming unit becomes joined to the 5 oxygen
atom of the growing chain to form a phosphite triester.

The 5 -OH group of the activated monomer is unreactive because it is blocked by a

dimethoxytrityl (DMT) protecting group, and the 3 -phosphoryl group is rendered
unreactive by attachment of the b -cyanoethyl ( b CE) group.

Likewise, amino groups on the purine and pyrimidine bases are blocked.

Coupling is carried out under anhydrous conditions because water reacts with
phosphoramidites.
In step 2, the phosphite triester (in which P is trivalent) is oxidized by iodine to form a
phosphotriester (in which P is pentavalent).

In step 3, the DMT protecting group on the 5 -OH of the growing chain is removed by
the addition of dichloro-acetic acid, which leaves other protecting groups intact.
The DNA chain is now elongated by one unit and ready for another cycle of
addition.
Each cycle takes only about 10 minutes and elongates more than 98% of the
chains.
This solid-phase approach is ideal for the synthesis of DNA, as it is for
polypeptides, because the desired product stays on the insoluble support until the
final release step.

All the reactions take place in a single vessel, and excess soluble reagents can
be added to drive reactions to completion.
At the end of each step, soluble reagents and by-products are washed away from
the glass beads that bear the growing chains.
At the end of the synthesis, NH3 is added to remove all protecting groups and
release the oligonucleotide from the solid support. Because elongation is never
100% complete, the new DNA chains are of diverse lengths the desired chain is
the longest one.

The sample can be purified by high pressure liquid chromatography or by

electrophoresis on polyacrylamide gels. DNA chains of as many as 100
nucleotides can be readily synthesized by this automated method.
A molecule with a net charge will move in an electric field. This phenomenon,
termed electrophoresis, offers a powerful means of separating proteins and
other macromolecules, such as DNA and RNA.

Electrophoretic separations are nearly always carried out in gels (or on solid
supports such as paper) because the gel serves as a molecular sieve that
enhances separation.

Molecules that are small compared with the pores in the gel readily move
through the gel, whereas molecules much larger than the pores are almost
immobile. Intermediate size molecules move through the gel with various
degrees of facility.
Electrophoresis is performed in a thin, vertical slab of polyacrylamide.

The direction of flow is from top to bottom. Polyacrylamide gels, formed by the
polymerization of acrylamide and cross-linked by methylenebisacrylamide,
are choice supporting media for electrophoresis because they are chemically
inert and are readily formed.
The ability to rapidly synthesize DNA chains of any selected sequence opens
many experimental avenues.

For example, synthesized oligonucleotide labeled at one end with 32P or a

fluorescent tag can be used to search for a complementary sequence in a very
long DNA molecule or even in a genome consisting of many chromosomes.

The use of labeled oligonucleotides as DNA probes is powerful For example, a

DNA probe that can base-pair to a known complementary sequence in a
chromosome can serve as the starting point. Such a probe can be used as a
primer to initiate the replication of neighboring DNA by DNA polymerase.

One of the most exciting applications of the solid-phase approach is the synthesis
of new tailor-made genes. New proteins with novel properties can now be
produced in abundance by expressing synthetic genes
Maxam–Gilbert sequencing was the first widely adopted method for DNA
sequencing, and, along with the Sanger dideoxy method, represents the
first generation of DNA sequencing methods

Maxam–Gilbert sequencing requires radioactive labeling at one 5′ end of the

DNA fragment to be sequenced (typically by a kinase reaction using gamma-
32
P ATP) and purification of the DNA.

Chemical treatment generates breaks at a small proportion of one or two of the

four nucleotide bases in each of four reactions (G, A+G, C, C+T).

For example, the purines (A+G) are depurinated using formic acid,
the guanines (and to some extent the adenines) are methylated by dimethyl
sulfate,

The pyrimidines (C+T) are hydrolysed using hydrazine. The addition of salt
(sodium chloride) to the hydrazine reaction inhibits the reaction of thymine for
the C-only reaction.
The modified DNAs may then be cleaved by hot piperidine; (CH2)5NH at the
position of the modified base.

The concentration of the modifying chemicals is controlled to introduce on

average one modification per DNA molecule.

Thus a series of labeled fragments is generated, from the radiolabeled end to

the first "cut" site in each molecule.

The fragments in the four reactions are electrophoresed side by side in

denaturing acrylamide gels for size separation.

To visualize the fragments, the gel is exposed to X-ray film

for autoradiography, yielding a series of dark bands each showing the
location of identical radiolabeled DNA molecules. From presence and
absence of certain fragments the sequence may be inferred.
Enzymes used in genetic engineering

Used for in vitro DNA synthesis.

Examples:
DNA polymerase I
Klenow fragment
T4 DNA polymerase
T7 DNA polymerase
Terminal deoxynucleotidyl transferase
Reverse transcriptase
•In replication process, RNase removes the RNA primer
(created by Primase) from the lagging strand and then
Polymerase I fills in the necessary nucleotides between
the Okazaki fragments in a 5'→3' direction, proofreading
for mistakes as it goes.

•TdT catalyses the addition of nucleotides to the 3'

terminus of a DNA molecule. Unlike most DNA
polymerases, it does not require a template. The preferred
substrate of this enzyme is a 3'-overhang, but it can also
add nucleotides to blunt or recessed 3' ends. Cobalt is a
necessary cofactor, however the enzyme catalyzes reaction
upon Mg and Mn administration in vitro.
•Exposure of DNA polymerase I to the protease subtilisin
cleaves the molecule into a small fragment, which have 5’-3'
exonuclease activity, and a large piece called Klenow fragment

•Klenow Fragment (3´→ 5´ exo-) is an N-terminal truncation of

DNA Polymerase I which retains polymerase activity, but has
lost the 5´→ 3´ exonuclease activity .

•T4 DNA Polymerase catalyzes the synthesis of DNA in the 5´→

3´ direction and requires the presence of template and primer.
This enzyme has a 3´→ 5´ exonuclease activity which is much
more active than that found in DNA Polymerase I (E. coli).
Unlike E. coli DNA Polymerase I, T4 DNA Polymerase does
not have a 5´→ 3´ exonuclease function. Best enzyme to create
blunt end.
•T7 DNA Polymerase catalyzes the replication of T7 phage DNA
during infection. The protein dimer has two catalytic activities:
DNA polymerase activity and strong 3´→ 5´ exonuclease (1,2,3).
The high fidelity and rapid extension rate of the enzyme make it
particularly useful in copying long stretches of DNA template.

•RNA polymerase
RNA polymerase makes a RNA copy of a DNA or RNA.
Used in vitro to produce RNA complementary to one strand of
gene of interest that has been placed immediately downstream
from the promoter.

Examples:
E. coli RNA polymerase
SP6, T3 and T7 RNA polymerases
•Alkaline phosphatase
•Alkaline phosphatase catalyzes the removal of 5’
phosphate groups from DNA and RNA.
Examples:
Bacterial alkaline phosphatase (BAP)
Calf intestinal phosphatase (CIP or CIAP)
Arctic shrimp alkaline phosphatase (SAP)

•Polynucleotide kinase
PNK catalyzes the transfer of a -phosphate from ATP to
the 5’ hydroxyl termini of polynucleotides of either DNA
or RNA.
•DNA ligase
•DNA ligase catalyzes the formation of a phosphodiester
bond between the 5' phosphate of one strand of DNA or
RNA and the 3' hydroxyl of another.
•The enzyme requires either ATP or NAD+, as cofactor
depending on the source to form high-energy
intermediates.

Examples:
T4 DNA ligase
E. coli DNA ligase
Taq DNA ligase
T4 RNA ligase
•Deoxyribonuclease
•Deoxyribonuclease is a type of nuclease, which
catalyzes the hydrolytic cleavage of phosphodiester
bonds of the DNA backbone.
•Various types of DNase, which differ in their
substrate specificities, chemical mechanisms and
biological functions.
Examples
DNase I
Shrimp DNase
Bal 31 nuclease
S1 nuclease
Mung bean endonuclease
Exodeoxyribonucleases
•Ribonuclease
•Ribonuclease is a group of nucleases that
catalyzes the hydrolysis of RNA into smaller
components.

Examples
RNase A
RNase H
RNase T1

•Phosphodiesterase I
•It hydrolyzes 5' mononucleotides from 3' hydroxy
terminated DNA and RNA.
-Agarase
•β-Agarase I digests agarose, releasing trapped DNA
and producing soluble carbohydrate molecules.
•It can be used to purify both large (> 50 kb) and
small (< 50 kb) fragments of DNA from gels.

•Uracil-DNA glycosylase
•Uracil-DNA glycosylase (UDG) excises uracil from
dU-containing DNA by cleaving the N-glycosidic bond
between the uracil base and the sugar without
disturbing the phosphodiester backbone.
•Proteinase K
•Proteinase K efficiently digests the RNases and DNases in
cell lysates which is useful in purifying high molecular
weight DNA and RNA from cells and tissues.

•Proteinase K (EC 3.4.21.64, protease K, endopeptidase K,

Tritirachium alkaline proteinase, Tritirachium album serine
proteinase, Tritirachium album proteinase K) is a broad-
spectrum serine protease. The enzyme was discovered in
1974 in extracts of the fungus Engyodontium album
(formerly Tritirachium album). Proteinase K is able to
digest hair (keratin), hence, the name "Proteinase K".
•Proteinase K is commonly used in molecular biology to digest
protein and remove contamination from preparations of nucleic
acid. Addition of Proteinase K to nucleic acid preparations rapidly
inactivates nucleases that might otherwise degrade the DNA or
RNA during purification since the enzyme is active in the presence
of chemicals that denature proteins, such as SDS and urea,
chelating agents such as EDTA, sulfhydryl reagents, as well as
trypsin or chymotrypsin inhibitors.

•Proteinase K is used for the destruction of proteins in cell lysates

(tissue, cell culture cells) and for the release of nucleic acids, since
it very effectively inactivates DNases and RNases.
•Lysozyme
•Lysozymes catalyzes the hydrolysis of 1, 4--
linkages between NAG and NAM acid residues of
peptidoglycan of bacterial cell walls and facilitates
DNA isolation.

•Topoisomerase
•This enzyme helps in relaxation of both positively
and negatively supercoiled DNA and used to enhance
the electrophoretic separation of plasmid DNAs.
 From the Greek - klon, a twig
 An aggregate of the asexually produced progeny of an
individual;a group of replicas of all or part of a
macromolecule (such as DNA or an antibody)
 An individual grown from a single somatic cell of its
parent & genetically identical to it

 Clone: a collection of molecules or cells, all identical to

an original molecule or cell
DNA CLONING

A method for identifying and purifying a

particular DNA fragment (clone) of interest
from a complex mixture of DNA fragments,
and then producing large numbers of the
fragment (clone) of interest.
 When DNA is
extracted from an
organism, all its
genes are obtained
 In gene (DNA)
cloning a
particular gene is
copied (cloned)
 A particular gene can be isolated and its
nucleotide sequence determined
 Control sequences of DNA can be identified &
analyzed
 Protein/enzyme/RNA function can be
investigated
 Mutations can be identified, e.g. gene defects
related to specific diseases
 Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production, insect
resistance, etc.
Sources of DNA for Cloning

 1) Chromosomal DNA

 2) RNA converted to cDNA

 3) PCR-amplified DNA
 Restriction endonucleases
 Ligase
 Vectors
 Host
 Methods for introducing DNA into a host
cell
 Restriction endonucleases
(restriction enzymes)
› sticky ends
› blunt ends

 Nomenclature
› EcoRI
› E = genus (Escherichia)
› co = species (coli)
› R = strain
› I = # of enzyme
Blunt & Sticky ends
 Complementary ends
(sticky ends) H-bond
 Ligase forms
phosphodiester bond
to seal strands
together.
Allowing the exogenous DNA to be inserted, stored, and
manipulated mainly at DNA level.

1 Plasmid vectors
2 Bacteriophage vectors
3 Cosmids
4 BACs & YACs
 insert size
 vector size

 restriction sites

 copy number

 cloning efficiency

 ability to screen for inserts

A vector that is used predominantly for
reproducing the DNA fragment is often referred
to as a cloning vector, while if it is used for
expressing a gene contained within the cloned
DNA, it is called an expression vector

Relaxed plasmids are maintained at multiple

copies per cell (10–200), while stringent plasmids
are present at a single copy, or a low number of
copies (1–2) per cell. In general, relaxed plasmids
replicate using host derived proteins, while
stringent plasmids encode protein factors that are
necessary for their own replication.
 Cloning vectors are DNA molecules that are used to
"transport" cloned sequences between biological hosts
and the test tube.

Cloning vectors share four common properties:

1. Ability to promote autonomous replication.
2. Contain a genetic marker (usually dominant) for
selection.
3. Unique restriction sites to facilitate cloning of insert
DNA.
4. Minimum amount of nonessential DNA to optimize
cloning.
 Involves five steps:
Enzyme restriction digest of DNA sample.
Enzyme restriction digest of DNA plasmid vector.
Ligation of DNA sample products and plasmid vector.
Transformation with the ligation products.
Growth on agar plates with selection for antibiotic
resistance.
 Plasmids are circular DNA
molecules present in the
cytoplasm of the bacteria
 Capable of autonomous
replication
 Can transfer genes from
one cell to other
 Act as vectors in genetic
engineering.
 Can also present in
Yeasts
 Some plasmids are present
in multiple copies in the cell
 Plasmid vectors are double-stranded, circular, self-
replicating, extra-chromosomal DNA molecules.

 Advantages:
› Small, easy to handle
› Straightforward selection strategies
› Useful for cloning small DNA fragments
(< 10kbp)
 Disadvantages:
› Less useful for cloning large DNA fragments
(> 10kbp)
1. Contains an origin of replication, allowing for
replication independent of host’s genome.
2. Contains Selective markers: Selection of cells
containing a plasmid
twin antibiotic resistance
blue-white screening
3. Contains a multiple cloning site (MCS)
4. Easy to be isolated from the host cell.
 Plasmid vectors are ≈1.2–
3kb and contain:
 replication origin (ORI)
sequence
 a gene that permits
selection,
 Here the selective gene is
ampr; it encodes the
enzyme b-lactamase, which
inactivates ampicillin.
 Exogenous DNA can be
inserted into the bracketed
region .
 Selective marker is required for
maintenance of plasmid in the cell.
 Because of the presence of the
selective marker the plasmid
becomes useful for the cell.
 Under the selective conditions, only
cells that contain plasmids with
selectable marker can survive
 Genes that confer resistance to
various antibiotics are used.
 Genes that make cells resistant to
ampicillin, neomycin, or
chloramphenicol are used
 Origin of replication is a
DNA segment recognized
by the cellular DNA-
replication enzymes.
 Without replication
origin, DNA cannot be
replicated in the cell.
 Many cloning vectors contain a
multiple cloning site or
polylinker: a DNA segment
with several unique sites for
restriction endo- nucleases
located next to each other
 Restriction sites of the polylinker
are not present anywhere else
in the plasmid.
 Cutting plasmids with one of the
restriction enzymes that
recognize a site in the polylinker
does not disrupt any of the
essential features of the vector
 Gene to be cloned can
be introduced into the
cloning vector at one
of the restriction sites
present in the
polylinker
pBR322 was a breakthrough for molecular biology as the first widely
used plasmid for molecular cloning, but the double screening
procedure required to identify recombinant clones was both time
consuming and error prone.

In 1982, a new series of plasmids were developed that permitted the

identification of the foreign DNA containing cells in a single screening
step. These are called the pUC plasmids (Vieira and Messing, 1982).
They have three important additional features compared with pBR322

High copy number – a mutation within the origin of replication produces

500–600 copies of the plasmid per cell without the need for
chloroamphenicol amplification.

The mutation, a G to A change one nucleotide upstream of the

initiation site of RNAI, reduces the level of the RNAI transcript and
consequently results in an increase in DNA replication using RNAII as
the primer.
Plasmid vectors
pBR322 carries the
ColE1 replication
origin and rop gene
to ensure reasonably
high plasmid copy
number (15–20
copies per cell),
which can be
increased 200-fold by
chloramphenicol
amplification
 lacZ lacZ insert

functional enzyme nonfunctional enzyme

X-gal product X-gal product
α-complementation and XGal
staining. XGal (5-bromo-4-chloro-3-
indolylβ-D-galactoside) is cleaved by
a functional β-galactosidase enzyme
into galactose and a 5-bromo-4-
chloro-indoxyl derivative. The indoxyl
spontaneously dimerizes and oxidizes
to form an insoluble blue dye.

The βgalactosidase enzyme is the

product of the lacZ gene and the
active form of the enzyme is a
tetramer of identical polypeptides.

Certain mutants of lacZ (such as

lacZM15) produce versions of the
protein that do not include the
extreme amino-terminal end of the
1173 amino acid polypeptide. Such
derivatives, termed the ω-peptide, are
unable to form the active tetramer
and are not functional as β-
galactosidase enzymes. The ω-
peptide can be made functional by
co-expressing the lacZ α-peptide
(amino acids 1–63) in the same cell.
The α-peptide promotes tetramer
formation and restores enzymatic
function
 Plasmid vectors are used
to clone DNA ranging in
size from several base
pairs to several thousands
of base pairs (100bp -
10kb).
 ColE1 based, pUC vehicles

commercially available
ones, eg pGEM3,
pBlueScript
 Cannot accept large fragments
 Sizes range from 0- 10 kb
 Standard methods of transformation
are inefficient
 Phage lambda is a bacteriophage or phage, i.e. bacterial
virus, that uses E. coli as host.
 Its structure is that of a typical phage: head, tail, tail fibres.
 Lambda viral genome: 48.5 kb linear DNA with a 12 base
ssDNA "sticky end" at both ends; these ends are
complementary in sequence and can hybridize to each other
(this is the cos site: cohesive ends).
 Infection: lambda tail fibres adsorb to a cell surface receptor,
the tail contracts, and the DNA is injected.
 The DNA circularizes at the cos site, and lambda begins its
life cycle in the E. coli host.
 Advantages:
› Useful for cloning large DNA fragments
(10 - 23 kbp)
› Inherent size selection for large inserts

 Disadvantages:
› Less easy to handle
 Left arm:
› head & tail proteins
 Right arm:
› DNA synthesis
› regulation
› host lysis
 Deleted central region:
› integration & excision
› regulation
 Bacteriophage lambda as a cloning vector

transduction: transfer of host genes from one cell to another by a virus

Upon infection, bacteriophage λ attaches
to the surface of a bacterial cell, and its
DNA enters the bacterium. Almost
immediately, the λ DNA circularizes. The
DNA can then enter either the lysogenic
or the lytic pathway. During lysogeny,
the λ DNA integrates into the E. coli
chromosome and is replicated, along with
the host DNA, such that the prophage is
passed onto subsequent generations. In
the lytic phase, the λ DNA does not
integrate, but is immediately replicated
and transcribed to produce new phage
particles. Eventually, bacterial cell lysis
occurs and the newly formed phage are
released into the surrounding medium.
The lysogenic prophage may be induced
into the lytic cycle by, for example,
treatment with UV light. In this case the λ
DNA loops out of the E. coli genome and
the lytic pathway is initiated
Lambda Insertion vector : first remove nonessential region
and introduce single restriction site
e.g. lambdagt10 (C1 gene contains res site EcoR1)
Lambda ZAPII (lacZ gene)

Replacement Vector : two restriction site

Lambda DASHII vector
Selection based on genome size and Spi phenotype
 Thus, have some advantages of Lambda as
Cloning Vehicle:
 Strong selection for cloning of large inserts
 Infection process rather than transformation for
entry of chimeric DNA into E. coli host
 Maintain Cosmids as phage particles in solution
 But Cosmids are Plasmids:
Thus do NOT form plaques but rather cloning
proceeds via E. coli colony formation
 Purpose:
1. Clone large inserts of
DNA: size ~ 45 kb
 Features:
Cosmids are Plasmids
with one or two Lambda
Cos sites.
 Presence of the Cos site
permits in vitro
packaging of cosmid
DNA into Lambda
particles
Combine the properties of plasmid vectors with the useful
properties of the cos site
 Advantages:
› Useful for cloning very large DNA fragments
(32 - 47 kbp)
› Inherent size selection for large inserts
› Handle like plasmids
 Disadvantages:
› Not easy to handle very large plasmids
› (~ 50 kbp)
The single-stranded M13 genome is
encased by coat proteins.
Bacterial infection occurs when the
phage particle attaches to the E. coli
pilus and the single DNA strand is
injected into the host.
The DNA is immediately converted to a
double-stranded form and is replicated
and transcribed to produce viral
proteins.
The build-up of viral protein 2
eventually forces asymmetric DNA
replication to produce single DNA
strands.
These are packaged into new viral
particles, which are secreted from the
bacteria without cell lysis occurring
M13 cloning vectors
have
been developed for
the selection of
DNA sequences
capable of
directing initiation
of DNA synthesis
on single-stranded
templates.
The DNA fragment encoding the target gene is transferred
from one plasmid to another by the action of the Cre
recombinase.

Cre is a 38 kDa recombinase protein from the bacteriophage

P1. It mediates recombination between or within DNA
sequences at specific locations called loxP sites.
These lox sites consist of two 13 bp inverted repeats
separated by an 8 bp spacer region.

The 8 bp spacer region in the loxP site has a defined

orientation that forces recombination to occur in a precise
direction and orientation.
The target gene, once transferred, will become linked to the specific
expression elements for which the acceptor vector has been
designed.

if the coding sequence for the gene of interest is in frame with the
upstream loxP site in the donor vector, it will automatically be in
frame with all peptides designed in the acceptor vector.

An alternative donor and acceptor plasmid system is based upon

site-specific recombination reactions mediated by phage λ. In this
case, DNA fragments flanked by recombination sites (att) can be
transferred into vectors containing compatible recombination sites
in a reaction mediated by the λ recombination proteins
The λ genomic DNA is injected into the cell and almost immediately
circularizes. At this point it can enter one of two pathways.

Lysogenic pathway. The phage DNA becomes integrated into the

bacterial genome (via homologous recombination between attP and
the bacterial genomic attB site) and is replicated along with the
bacterial DNA.

The prophage DNA remains integrated until it is induced to enter the

lytic pathway.

• Large-scale production of bacteriophage particles (proteins and

DNA) occurs that eventually leads to the lysis of the cell. The decision
as to whether lysis or lysogeny occurs is the result of the activity of
the cII protein. Active cII is required for the transcription of the cI
repressor
Bacterial artificial chromosomes (BAC) were developed by Mel
Simmons and coworkers in the early 1990s It is based on the
fertility factor (F factor) of Escherichia coli.

The F plasmid, a ~ 100 kb circular double stranded DNA, is

present is an E. coli cell in only 1-2 copies. The synthetic BAC
vectors, which are only ~7.5 kb double stranded DNA circles
contain the replication origin oriS and the gene repE of the F
plasmid that are responsible for initiation and proper orientation
of replication of the BAC vector.

The parA and parB genes of the F plasmid ensure efficient

segregation of the F factor into the daughter E. coli cells after
its replication are also incorporated in the BAC vector. The BAC
vectors also contain multiple cloning sites (mcs), a selectable
marker in the form of antibiotic resistance and colour based
identification (lac Z complementation system) of recombinants
carrying inserts.
The naturally occurring F2 factors consist of up to 25% of the E. coli
genome integrated into the basic F factor and are very stable. This
 oriS and oriE mediate replication
 parA and parB maintain single
copy number
 ChloramphenicolR marker
 The components of partition
systems are commonly
named ParA (the partition
ATPase), ParB (the
centromere-binding protein),
and parS (the centromere or
partition site). Plasmids
typically contain one parS located
near the parA and parB genes,
although some have multiple
parS
 Purpose:
 Cloning vehicles that propogate in eukaryotic cell
hosts as eukaryotic Chromosomes
 Clone very large inserts of DNA: 100 kb - 10 Mb
 Features:
YAC cloning vehicles are plasmids
Final chimeric DNA is a linear DNA molecule with
telomeric ends
 Additional features:
 Often have a selection for an insert
 YAC cloning vehicles often have a bacterial
origin of DNA replication (ori) and a selection
marker for propogation of the YAC through
bacteria.
 The YAC can use both yeast and bacteria as a
host
 Retroviral vectors are used to introduce new or altered
genes into the genomes of human and animal cells.
 Retroviruses are RNA viruses.
 The viral RNA is converted into DNA by the viral reverse
transcriptase and then is efficiently integrated into the
host genome
 Any foreign or mutated host gene introduced into the
retroviral genome will be integrated into the host
chromosome and can reside there practically
indefinitely.
 Retroviral vectors are widely used to study oncogenes
and other human genes.
Yeast artificial chromosome (YAC) is a human-engineered
DNA molecule used to clone DNA sequences in yeast cells.
YACs are often used in connection with the mapping and
sequencing of genomes. Segments of an organism's DNA, up to
one million base pairs in length, can be inserted into YACs.

YAC vectors have a high cloning capacity as its insert can be

up to 200kb-2000kb in size
 large
inserts

ARS URA3 HIS3

telomere centromere markers telomere
replication
origin
 BACs : Bacterial Artificial Chromosomes
YACs : Yeast Artificial Chromosomes
 Advantages:
› Useful for cloning extremely large DNA fragments
(100 - 2,000 kbp)
› This is very important for genome sequencing
projects

 Disadvantages:
› Not easy to handle extremely large DNA molecules