MOLECULAR

BIOTECHNOLOGY
 CONTENT

2.0 Recombinant DNA Technologies

2.1 Restriction endonucleases
2.2 Cloning Vectors
2.3 Transformation and Selection
2.4 Regulation of gene expression in
prokaryotes
2.5 Constructing and screening gene
libraries
 MOLECULAR
BIOTECHNOLOGY
• Recombinant DNA Technology:
– Insertion or modification of genes to produce
desired proteins
• Genetic engineering:
- manipulation of genes /insert DNA into cells
• Gene Cloning:
- isolating genes from one organism,
manipulating purified DNA in vitro, and
transferring to another organism
MOLECULAR BIOTECHNOLOGY
It all began with
• Arber (1950)-discovered enzymes that
degrade bacterial viruses

 1953 - double helical structure of DNA published in

Nature by Watson and Crick*

• Smith (1970)-purified the enzymes and

characterized them
– Cut DNA at specific sites
– Called restriction enzymes
1973: Cohen and Boyer
• created the first recombinant DNA using
bacterial genes
• 1980 - the U.S. patent for cloning genes is
awarded to Cohen and Boyer**
 First biotech companies formed:
1976 - Genentech
1978 - Biogen
1980 - Amgen
The industry is
1981 - Immunex
only 25 years old…
1981 - Chiron
it takes time!
1981 - Genzyme
*Nature 171, 737(April 2, 1953)
**Proc Natl Acad Sci U S A. 1973 Nov;70(11):3240-4 and
Proc Natl Acad Sci U S A. 1974 May;71(5):1743-7
 Recombinant DNA

• Production of a unique DNA molecule by

joining together two or more DNA
fragments not normally associated with
each other
• DNA fragments are usually derived from
different biological sources
 Importance of Recombinant DNA
Technology

1. Recombinant DNA technology have revolutionalized the

search for defective genes that cause human diseases
such as cystic-fibrosis, thalassaemia and sickele-cell
anaemia.

2. Mutation responsible for the diseases have been

determined by comparing the nucleotide sequences of
wild type and mutant alleles of the genes. The results
allows treatment of diseases by gene therapy.
3. Genetic engineering is finding wide uses in medicine,
industry and agriculture.
Valuable proteins that could be isolated from eukaryotes
only in a small amounts and at a great expense now can be
produced in a large quantities in genetically engineered
bacteria.
For example, human insulin and growth hormone. Plants
may be made resistant to insect attack.
Genetic engineering techniques are also used for diagnosis
of inherited disorders and in forensic medicine in the
identification of related individuals.
 Basic Events in Gene Cloning
• Isolation of gene of interest
• Incorporate gene into a vector
• Introduce recombinant vector into host cell
via transformation
• Select for the cells that have acquired the
recombinant DNA molecule
• Multiply recombinant vector within host cell to
produce a number of identical copies of the
cloned gene
 Components of Gene Cloning

• Vectors (cloning vehicles)

• Enzymes for cutting and joining the
DNA fragments
• The DNA fragments (libraries)
• Selection process
CLONING VECTORS
 CLONING VECTORS
• 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.
 Types of Vectors

• Plasmids
• Cosmids
• Phages
• Yeast Artificial Chromosomes
(YACs)
• Bacterial Artificial Chromosomes
(BACs)
 Plasmids
• Double stranded, circular DNA which exist in
bacteria, yeast and organelles
• May exist as single copy per cell or multi-
copy
per cell (10-20 genomes/cell), or even under
relaxed replication control where up to 1000
copies/cell can be maintained
• Size of rDNA insertions limited to ~10kb
 PLASMIDS
• Bacterial cells may
contain extra-
chromosomal DNA
called plasmids.
• Plasmids are usually
represented by small,
circular DNA.
• Some plasmids are
present in multiple
copies in the cell
 PLASMID VECTORS
Plasmid vectors are ≈1.2–3kb
and contain:
• replication origin (ORI)
• 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
• 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
• 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
 MULTIPLE CLONING SITE

• Gene to be cloned can

be introduced into the
cloning vector at one of
the restriction sites
present in the polylinker
A typical plasmid DNA might have
the following features
Disadvantages using plasmids

• Cannot accept large fragments

• Sizes range from 0- 10 kb
• Standard methods of
transformation are inefficient
 CLONING VECTORS
• Different types of cloning vectors are
used for different types of cloning
experiments.
• The vector is chosen according to the size
and type of DNA to be cloned
 BACTERIOPHAGE LAMBDA
• 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.
 Bacteriophage Vectors
• Viruses that attack bacteria
• Must first deactivate lysogenic growth
component of phage (phage DNA inserts into host
DNA, creating prophage)
• Allow lytic growth – cell death after infection and
replication. Cell death revealed as plaques
• Insert rDNA into phage (usu. up to 25kb)
• Infect bacteria with phage
• Infected bacteria form plaques
• Advantage: Transformation, selection very easy
BACTERIOPHAGE LAMBDA
 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
 Cosmids

• Plasmid vectors that contain a

bacteriophage lamda cos site
• The cos site results in efficient
packaging of lamda DNA into virus
particles
• So, with the cos site, larger DNA inserts
are possible (up to ~40 kb)
 COSMID VECTOR
• 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

• Cosmids are Plasmids:
Thus do NOT form plaques but rather cloning
proceeds via E. coli colony formation
Yeast Artificial Chromosomes
 Yeast Artificial Chromosomes
• 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: Artificial Chromosome
• 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
 BAC
• BACs - Bacterial Artificial Chromosomes

• These chimeric DNA molecules use a naturally-

occurring low-copy number bacterial plasmid origin of
replication, such as that of F-plasmid in E. coli.

• Can be cloned as a plasmid in a bacterial host, and

its natural stability generally permits cloning of large
pieces of insert DNA, i.e. up to a few hundred kb of
DNA
SELECTION AND SCREENING
• Selection refers to application of conditions
that favors the growth of cells or phages that
carry the vector or vector and insert.
• Screening allows all cells to grow, but tests
the resulting clones for the presence of the
insert in the vector.
 BLUE/WHITE SCREENING
• Colony Selection: finding the rare
bacterium with recombinant DNA

• Only E. coli cells with resistant plasmids

grow on antibiotic medium
• Only plasmids with functional lacZ gene
can grow on Xgal
 lacZ(+) => blue colonies
lacZ functional => polylinker intact =>
nothing inserted, no clone
lacZ(-) => white colonies polylinker
disrupted => successful insertion &
recombination!
 α -complementation
• The portion of the lacZ gene encoding the first
146 amino acids (the α -fragment) are on the
plasmid
• The remainder of the lacZ gene is found on the
chromosome of the host.
• If the α -fragment of the lacZ gene on the plasmid
is intact (that is, you have a non-recombinant
plasmid), these two fragments of the lacZ gene
(one on the plasmid and the other on the
chromosome) complement each other and will
produce a functional β -galactosidase enzyme.
 SCREENING RECOMBINANTS

• In the example shown above, the b-galactosidase gene is

inactivated. The substrate "X-gal" turns blue if the gene is
intact, ie. makes active enzyme. White colonies in X-gal
imply the presence of recombinant DNA in the plasmid.
II. Restriction Endonucleases
 A. Origin and function

• First discovered in the late 1960s

• Bacterial origin
• Cleave foreign DNA to prevent invasion

• Named after the organism from which

they were derived
– EcoRI from Escherichia coli
– BamHI from Bacillus amyloliquefaciens
• Protect bacteria from bacteriophage infection
– Restricts viral replication
• Bacterium protects it’s own DNA by
methylating those specific sequence motifs
 Enzymes are the Tools of
DNA Technology

• These enzymes are called Restriction Endonucleases

• Restriction - Because for the way they work, they restrict

virus to only one host bacterial strain. They are also
restricted to acting on only specific DNA sequences

• Endonuclease - They cut nucleic acids in the middle not

just the ends
MOLECULAR SCISSORS

Restriction enzymes are

molecular scissors
 RESTRICTION ENZYMES

• A restriction enzyme cuts double-

stranded or single stranded DNA at
specific recognition nucleotide
sequences known as restriction sites.
• While recognition sequences vary widely ,
with lengths between 4 and 8 nucleotides,
many of them are palindromic.
PALINDROMES IN DNA
SEQUENCES
Genetic palindromes are
similar to verbal palindromes.
A palindromic sequence in
DNA is one in which the 5’ to
3’ base pair sequence is
identical on both strands (the
5’ and 3’ ends refers to the
chemical structure of the
DNA).
 NOMENCLATURE OF RESTRICTION
ENZYME
• Each enzyme is named after the bacterium from which
it was isolated using a naming system based on bacterial
genus, species and strain.
For e.g EcoRI
 Titles of restriction enzymes are derived from
first letter of the genus
+
first two letters of the species
. EcoRI -  from Escherichia coli
BamHI - from Bacillus amyloliquefaciens
HindIII - from Haemophilus influenzae
PstI -  from Providencia stuartii
Sau3AI - from Staphylococcus aureus
AvaI -  from Anabaena variabilis
 The names for restriction enzymes come from:

• the type of bacteria in which the enzyme is found

• the order in which the restriction enzyme was
identified and isolated.

EcoRI for example

R strain of E.coli bacteria
I as it is was the first E. coli restriction enzyme to
be discovered.
 Derivation of the EcoRI name

Abbreviation Meaning Description

E Escherichia genus

co coli species

R RY13 strain

order of identification
I First identified
in the bacterium
 B. Availability

• Over 200 enzymes have been identified,

and many are available commercially
from biotechnology companies
TYPES OF RESTRICTION ENZYMES

• Restriction endonucleases are

categorized into three general groups.
– Type I
– Type II
– Type III
 C. Classes

• Type I
– Cuts the DNA on both strands but at a non-
specific location at varying distances from
the particular sequence that is recognized by
the restriction enzyme
– Therefore random/imprecise cuts
– Not very useful for rDNA applications
• Type II
– Cuts both strands of DNA within the
particular sequence recognized by the
restriction enzyme
– Used widely for molecular biology
procedures
– DNA sequence = symmetrical
• Reads the same in the 5’ 3’ direction on
both strands = Palindromic Sequence
• Some enzymes generate “blunt ends” (cut in
middle)
• Others generate “sticky ends” (staggered
cuts)
– H-bonding possible with complementary tails
– DNA ligase covalently links the two fragments together
by forming phosphodiester bonds of the phosphate-
sugar backbones
 Hae III
HaeIII is a restriction enzyme that searches the DNA
molecule until it finds this sequence of four nitrogen
bases.

5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
Once the recognition site is found Hae III will cleave
the DNA at that site

5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
 These cuts produce
“blunt ends”

5’ TGACGGGTTCGAGG CCAG 3’
3’ ACTGCCCAAGGTCC GGTC 5’
 “blunt ends” and “sticky ends”
Hae III produced a “blunt end”?
EcoRI makes a staggered cut and produces a
“sticky end”
5’ GAATTC 3’
3’ CTTAAG 5’
5’ GAATTC 3’
3’ CTTAAG 5’
5’ G AATTC 3’
3’ CTTAA G 5’
More examples of restriction sites of restriction
enzymes with their cut sites

Hind III: 5’ AAGCTT 3’

3’ TTCGAA 5’

Bam HI: 5’ GGATCC 3’

3’ CCTAGG 5’

Alu I: 5’ AGCT 3’
3’ TCGA 5’
• Some recognise very short sequences
consisting of only 4 base pairs. These
tend to cut DNA more frequently
(generating smaller fragments)
• Some require longer recognition
sequences (up to 8 bp). The longer
the recognition sequence the less
frequently these sites are likely to
occur
DNA LIBRARIES
 Screening DNA Libraries
• Probe hybridization screens
– Using antibody probes to detect antigenic
fusion proteins -
• Protein-specific antibodies can be used as
"probes" to immunologically detect bacterial
fusion proteins encoding the
protein antigen.
Vectors such as λgt11 and lambda Zap
phage can be used.