Recombinant DNA Technology

Principles and methods of recombinant DNA technology

Molecular Cloning

Tools used in Recombinant DNA technology

Types of cloning vectors

General idea on cloning

Recombinant DNA Technology
• Methods for isolating, manipulating, and
amplifying identifiable DNA sequences.

• Allows us to study the structure and

function of individual genes.

• Allows for the directed genetic

manipulation of organism (modify gene
function, insert novel genes)
What is Cloning ?
• Clone: a collection of molecules or cells, all
identical to an original molecule or cell.
• To "clone a gene" is to make many copies of
it.
• A cloned gene can be an exact copy of a
natural gene.
• A cloned gene can be an altered version of a
natural gene.
• Recombinant DNA technology allows for in
vitro manipulation of a individual gene.
 Tools Needed for Cloning
• Source of DNA to be manipulated (PCR amplified
product, cDNA or genomic library)

• Vectors e.g Plasmids (where the gene of interest to be

inserted or placed)

• Restriction enzymes (molecular scissors)

• DNA ligase (paste)

• Suitable host e.g E. coli, yeast, human cell culture,

plant cell (biological machine needed to amplify
DNA)
Enzymes commonly used in molecular biology
Removes phosphate groups from 5' ends
Alkaline phosphatase of DNA (prevents unwanted re-ligation of
cut DNA)
Joins compatible ends of DNA fragments
DNA ligase (blunt/blunt or complementary cohesive
ends). Uses ATP
Synthesises DNA complementary to a
DNA template in the 5'-to-3'direction.
DNA polymerase
Starts from an oligonucleotide primer
with a 3' OH end
Digests nucleotides progressiviely from a
Exonuclease III
DNA strand in the 3' -to-5' direction
Adds a phosphate group to the 5' end of
Polynucleotide kinase double- or single-stranded DNA or RNA.
Uses ATP
RNase A Nuclease which digests RNA, not DNA
Heat-stable DNA polymerase isolated
Taq polymerase from a thermostable microbe (Thermus
aquaticus)
Restriction Enzymes
What are restriction enzymes?

• Molecular scissors that cut double stranded

DNA molecules at specific points.

• Found naturally in a wide variety of

prokaryotes

• An important tool for manipulating DNA.

Discovery
• Arbor and Dussoix in 1962 discovered that certain
bacteria contain endonucleases which have the ability
to specifically cleave DNA.

• In 1970 Smith and colleagues purified and

characterized the cleavage site of a Restriction
Enzyme.

• Werner Arbor, Hamilton Smith and Daniel Nathans

shared the 1978 Nobel prize for Medicine and
Physiology for their discovery of Restriction Enzymes.
 Types of Restriction Enzymes
Cleavage Location of Examples
site methylase

Type I Cause random Endonuclease EcoK I

cleavage, and methylase EcoA I
around 1000bp activities located CfrA I
away from on a single
recognition site protein molecule
Type II Very specific, Endonuclease EcoR I
cut the DNA and methylase BamH I
within the activities are Hind III
recognition site located in
separate units
Type III Cause random Endonuclease EcoP I
cleavage, and methylase Hinf III
located on a
 Restriction fragments can be blunt ended or
sticky ended

5’ G A A T T C 3’ 5’ G A T A T C 3’
3’ C T T A A G 5’ 3’ C T A T A G 5’

Sticky or cohesive Blunt Ends

Ends

Sticky ends or blunt ends can be used to join DNA fragments.

Sticky ends are more cohesive compared to blunt ends.

 5’ ATGCGAATTCCGGTT 3’
3’ TACGCTTAAGGCCAA 5’
Sticky-end cutter
EcoR1

5’-ATGCG-3’ 5’-AATTCCGGTT-3’
3’-TACGCTTAA-5’ 3’-GGCCAA-5’

5’ ATGCGATATCCGGTT 3’
3’ TACGCTATAGGCCAA 5’
Blunt-end cutter
EcoRV

5’-ATGCGAT-3’ 5’-ATCCGGTT-3’
3’-TACGCTA-5’ 3’-TAGGCCAA-5’
 Stick ends
5’ Overhangs

• 5' overhangs: The enzyme cuts asymmetrically

within the recognition site such that a short single-
stranded segment extends from the 5' ends. Bam HI
cuts in this manner – Sticky end cutter.
3’ Overhangs

• 3' overhangs: Again, we see asymmetrical cutting

within the recognition site, but the result is a
single-stranded overhang from the two 3' ends.
KpnI cuts in this manner – blunt end cutter
Blunt ends

• Blunts: Enzymes that cut at precisely opposite

sites in the two strands of DNA generate blunt
ends without overhangs. SmaI is an example of an
enzyme that generates blunt ends.
 Isoschizomers and Neochizomers

• Restriction enzymes that have the same recognition sequence as

well as the same cleavage site are known as Isoschizomers.
e.g. SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers
of each other

• Restriction enzymes that have the same recognition sequence

but cleave the DNA at a different site within that sequence are
known asNeochizomers. Eg: SmaI and XmaI

CCCGGG CCCGGG
GGGCCC GGGCCC
Xma I Sma I
Biological function

• Most bacteria use Restriction Enzymes as a

defense against bacteriophages.

• Restriction enzymes prevent the replication of the

phage by cleaving its DNA at specific sites.

• The host DNA is protected by Methylases which

add methyl groups to adenine or cytosine bases
within the recognition site thereby modifying the
site and protecting the DNA.
Restriction Enzymes
Restriction/modification system (R/M)
• Bacteria protect themselves from attack by viruses
and other bacteria using a restriction/modification
system.
• Allows bacteria to recognize and destroy foreign
DNA
• Bacteria contain restriction endonucleases that
recognize and cleave specific sequences when they
are not methylated
• Bacteria contain DNA methylases that modify their
chromosomal DNA at specific sequences, protecting
from cleavage by restriction endonucleases
Restriction Modification System

AAGATGCGAATTCGTACA AAGATGCGAATTCGTACA

DNA methylase DNA methylase

* *
AAGATGCGAATTCGTACA AAGATGCGAATTCGTACA

Restriction endonuclease Restriction endonuclease

* *
AAGATGCGAATTCGTACA AAGATGCG AATTCGTACA
The Dam and Dcm methylases of E.coli
Most E.coli strains contain three site specific DNA methylases.

The Dam methylase, encoded by dam gene, transfer methyl group from S-adenosyl
Methionine (SAM) to N6 position of Adenine residue in the sequence 5’ GATC 3’

The Dcm methylase (also known as Mec methylase) encoded by dcm gene, methylates
the internal Cytosine residue at the C5 position within the sequences 5’CCAGG 3’ and
5’ CCTGG 3’.

Considering 50% GC content in E.coli genome, the recognition sites for Dam and Dcm
Methylases will occur, on average, after every 256-512 bp.

Sometimes a specific restriction endonuclease may become resistant to cleavage for

its respective recognition sequence in E.coli strains expressing the Dam or Dcm
Methylases.

In such case, particular base within the recognition sequence of the restriction
endonuclease becomes methylated when the Methylase recognition site overlaps with
Restriction Endonuclease recognition sequence.
 Number and size of restriction fragments
• Restriction enzymes can recognize specific 4 base, 6 base, 8
base sequences – the recognition sequences called palindromes
because of similarity with the word that read same backwords
as forwards

• The number and size of restriction fragments generated by a

restriction enzyme will depends on the frequency of the target
site in the DNA molecule

• Assuming 50% G+C content and random distribution of 4

bases –
• A 4 base recognition sequence will occur (44) every ~256 base
pairs
• A 6-base recognition site occurs every (46) 4096 bp,
• A 8-base recognition site occurs ((48) 65,536 bp
 DNA Ligase

DNA ligase is a specific type of enzyme

which facilitates the joining of DNA strands
together by catalyzing the formation of a
phosphodiester bond between the 5’ P and 3’
OH gr. of the broken DNA ends.
E. coli DNA ligase
The E. coli DNA ligase is encoded by the lig gene. DNA ligase in E. coli,
as well as most prokaryotes, uses energy gained by cleaving
nicotinamide adenine dinucleotide (NAD) to create the phosphodiester
bond. It does not ligate blunt-ended DNA except under conditions of
molecular crowding with polyethylene glycol, and cannot join RNA to
DNA efficiently.

T4 DNA ligase
The DNA ligase from bacteriophage T4 is the ligase most-
commonly used in laboratory research. It can ligate cohesive or
"sticky" ends of DNA, oligonucleotides, as well as RNA and RNA-
DNA hybrids, but not single-stranded nucleic acids. It can also
ligate blunt-ended DNA with much greater efficiency than E. coli
DNA ligase.
Unlike E. coli DNA ligase, T4 DNA ligase cannot utilize NAD and
it has an absolute requirement for ATP as a cofactor.
 Three different ligases in mammals
DNA ligase I: ligates the nascent DNA of the lagging strand after the
Ribonuclease H has removed the RNA primer from the Okazaki
fragments.

DNA ligase III: complexes with DNA repair protein XRCC1 to aid in
sealing DNA during the process of nucleotide excision repair and
recombinant fragments. Of the all known mammalian DNA ligases,
only Lig III has been found to be present in mitochondria.

DNA ligase IV: complexes with XRCC4. It catalyzes the final step in
the non-homologous end joining DNA double-strand break repair
pathway. It is also required for V(D)J recombination, the process that
generates diversity in immunoglobulin and T-cell receptor loci during
immune system development.

Plants: Ligase I, Ligase IV and a unique Ligase VI which is found only

in plants - required for rapid seed germination under optimal conditions
 Cloning Vectors
• Cloning vectors are DNA molecules that are used to
"transport" cloned sequences from the experimental
system to the biological host.

Cloning vectors share four common properties:

1. Ability to promote autonomous replication.

2. Unique restriction sites to facilitate cloning of insert
DNA, Multiple cloning sites (MCS)

3. Contain a genetic marker (usually dominant) for

selection of transformed cells containing clones.
4. Minimum amount of nonessential DNA to optimize
cloning.
Cloning Vector

Required features
1. Origin of replication
2. Selectable marker
3. Screenable marker for
recombinant
molecules
4. Cloning sites
 Selective markers

• 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

• Origin of replication is a DNA segment recognized

by the cellular DNA-replication enzymes.

• Without replication origin, DNA cannot be replicated

in the cell.
Multiple cloning site (MCS)
• 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
Types of cloning vectors

1. Plasmid cloning vectors

2. Bacteriophage based vectors

3. Cosmid vectors

4. Phagemid vectors

5. Yeast artificial chromosomes

6. PACs - P1-derived Artificial Chromosomes

7. BACs - Bacterial Artificial Chromosomes

Cloning Vehicles - Plasmids
• Naturally occurring extrachromosomal
DNA
• Self replicating circular double stranded
DNA molecules that have their own origin
of replication
• Usually present in multiple copies per cell
• Plasmids can be cleaved by restriction
enzymes, leaving blunt or sticky ends
• Circular plasmids can be reconstructed by
linking new DNA fragments to the blunt or
sticky ends of plasmid
 Plasmid vectors
• Plasmid vectors are ~1.5 - 4 kb
in size and contain:
• replication origin (ORI)
sequence
• An antibiotic resistant marker
gene that permits selection of
recombinant cells
• Here the selective gene is ampr;
it encodes the enzyme beta-
lactamase, which inactivates
ampicillin.
• Exogenous DNA can be inserted
into the specific site called
Multiple cloning sites,
containing recognition
sequences of several restriction
endonucleases.
Plasmid vectors

• Plasmid vectors are used to clone DNA

ranging in size from several base pairs
to several thousands of base pairs
Cloning capacity: 100 bp-5/10 kb
• ColE1 based, pUC series of cloning
vectors, commercially available are:
pGEM3, pBlueScript (pUC – p =
plasmid, UC – Univ. of California)
ColE1 is a plasmid found in bacteria. Its name derives from the fact that it
carries a gene for colicin E1 (the cea gene).
pUC18 is a plasmid cloning vector commonly used with E. coli. The
vector length is 2.6 kp and is isolated from E. coli strain DH5α by
standard procedures.
pBR322
pBR322 is a plasmid vector and one of the first widely used
cloning vector for E.coli

It was created in 1977 in the lab of Prof. Herbert Boyer at the

University of California

It was named after first names of two researchers, Bolivar and

Rodriguez, who constructed this plasmid vector.

4.3 kb in size, contain Ampicillin and tetracyclin resistant genes.

Contains Orc, and rop gene which encodes a protein that

regulates the plasmid copy number within the host bacteria.

The MCS contain restriction sequences of more than 40

restriction endonucleases.
 Bacteriophage 

• 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.
The virus codes for its own DNA polymerase which leads to the
synthesis of several hundred copies of viral DNA.

Over 100 different lambda vectors have been prepared for use
in cloning. The DNA of the cloning vector must be the correct
size for it to be packaged into virus particles (between 38 and
51kb).

The vector lambda gt10 (43.8 kbases) has an EcoRI site

inserted within the cI gene (lambda repressor) and so could
accept a DNA fragment of 7.6 kb before becoming too large to
be packaged into virus particles.

Other lambda vectors can incorporate DNA fragments of up to

22 kbases (e.g., the Charon series).

Cloning capacity: 5-25 kb

 Cosmid Vector

• 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
Cosmid vectors

• Thus, Cosmids have some advantages of

Lambda as Cloning Vehicle:
• Strongly selected 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
Phagemids
* Contains ori of filamentous phage f1, lacZ’ gene and MCS.
DNA can be cloned in a single-strand form via the help with
f1 helper phage
•contains T7 and T3 phage promoter for phage RNA
polymerase (in vitro transcription to produce RNA transcripts)

Helper phage:
a virus which helps a separate and
unrelated defective virus to reproduce by
infecting the same host cell that is already
occupied by the defective virus and
providing the proteins which are missing
in the defective virus to complete its life
cycle.
Yeast Artificial Chromosomes
Yeast Artificial Chromosomes

• Purpose:
• Cloning vehicles that propagate in
eukaryotic host cell as eukaryotic
Chromosomes
• Clone very large inserts of DNA: 100 kb -
10 Mb
• Features:
YAC cloning vehicles are like plasmids but
final chimeric DNA is a linear DNA
molecule with telomeric ends: Artificial
Chromosome
 YACs
• Additional features:

• YAC cloning vehicles often have a

bacterial origin of DNA replication (ori)
and a selection marker for propagation
of the YAC through bacteria.

• The YAC can use both yeast and bacteria

as a host
PACs and BACs

Phage P1-derived Artificial Chromosomes (PACs)

P1 is a temperate bacteriophage which has been extensively used for
Genetic analysis in E.coli as it mediates generalised transduction.

Sternberg and co-workers have developed P1 based vector – PACs with

the capacity of cloning large DNA fragments up to 100 kb.
This capacity is about 2-times more than cosmid vector but less than YAC.

PACs are useful for in vitro packaging of recombinant molecules into

Phage particles.

These vectors contain two loxP sites, which are recognized by phage
recombinase (product of cre gene) – leading to the circularization of
packaged
DNA after it has been injected into E.coli host.

The clones in PACs are maintained as low copy number plasmids by

selection for a vector kanamycin resistance marker.
 PACs and BACs
A bacterial artificial chromosome (BAC) is an engineered DNA
molecule used to clone DNA sequences in bacterial cells (for example,
E. coli).

BACs are often used in connection with DNA sequencing.

Segments of an organism's DNA, ranging from 100,000 to about

300,000 base pairs, can be inserted into BACs.

The BAC (bacterial artificial chromosome) system is based on a single copy

Sex factor or F factor of E.coli.

This vector includes the

 cos (cohesive) site,
 P1 derived loxP sites,
two cloning sites (HindIII and BamHI) and
several other restriction enzyme sites for
excision of the insert DNA.
The cloning site is flanked by T7
and SP6 promoters

The BACs can be transformed

easily in E.coli host cells

BACs are capable of carrying more

than 300 kb genomic DNA fragment
from human or plant genome.

Two widely used BAC vectors are

pBeloBAC11 and pECBAC1.
The fertility factor (first named F by one of its
discoverers Esther Lederberg; also called the sex factor in
E. coli or the F sex factor; also called F-plasmid) allows
genes to be transferred from one bacterium carrying the
factor to another bacterium lacking the factor by
conjugation.

The F factor was the first plasmid to be discovered. Unlike

other plasmids, F factor is constitutive for transfer proteins
due to a mutation in the gene finO.

The F plasmid belongs to a class of conjugative plasmids

that control sexual functions of bacteria with a fertility
inhibition (Fin) system.
F-primes are formed from Hfr's (high frequency
recombinations) by illegitimate recombination (i.e.
recombination between sequences with little
sequence homology)
 PACs and BACs
• PACs - P1-derived Artificial • BACs - Bacterial Artificial
Chromosomes Chromosomes
• E. coli bacteriophage P1 is • These chimeric DNA
similar to phage lambda in molecules use a naturally-
that it can exist in E. coli in a
prophage state. occurring low-copy
• Exists in the E. coli cell as a number bacterial plasmid
plasmid, NOT integrated into origin of replication, such
the E. coli chromosome. as that of F-plasmid in E.
• P1 cloning vehicles have been coli.
constructed that permit • Can be cloned as a
cloning of large DNA plasmid in a bacterial
fragments- few hundred kb of host, and its natural
DNA stability generally permits
• Cloning and propagation of cloning of large pieces of
the chimeric DNA as a P1 insert DNA, i.e. up to a
plasmid inside E. coli cells few hundred kb of DNA.
Retroviral vectors

• 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.
Shuttle Vector
A shuttle vector is a vector (usually a plasmid) constructed so that it can
propagate in two different host species.
DNA inserted into a shuttle vector can be manipulated in two different cell
types.
Shuttle vectors include plasmids that can propagate in eukaryotes and
prokaryotes (e.g. both Saccharomyces cerevisiae and Escherichia coli) or in
different species of bacteria (e.g. both E. coli and Rhodococcus erythropolis).
One of the most common types of shuttle vectors is the yeast shuttle vector.

Yeast shuttle vectors have components that allow for replication and selection
in both E. coli cells and yeast cells.

The E. coli component of a yeast shuttle vector includes an origin of replication

and a selectable marker, e.g. antibiotic resistance gene (e.g. beta lactamase for
Amp).

The yeast component of a yeast shuttle vector includes an autonomously

replicating sequence (ARS), a yeast centromere (CEN), and a yeast selectable
marker (e.g. URA3, a gene that encodes an enzyme for uracil synthesis
 Choice of cloning vectors

Maximum DNA insert possible with different cloning vectors

Victor Host Insert size

Plasmid E. Coli 5-10 kb

Bacteriophage  E. Coli 5-25 kb

Cosmids E. Coli 35-45 kb

P1 phage vector E. Coli 70-100 kb

PACs E. Coli 100-300 kb

BACs E. Coli ~300 kb

YACs Yeast 200 – 2000 kb