Recombinant DNA

Technology
GENE CLONING
 TERMS USED IN CLONING
DNA recombination.
The DNA fragment to be cloned is inserted into a
vector.
Transformation.
The recombinant DNA enters into the host cell and
proliferates.
Selective amplification.
A specific antibiotic is added to kill E. coli without
any protection. The transformed E. coli is protected
by the antibiotic-resistance gene
Isolation of desired DNA clones
 DNA CLONING
 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
To obtain large amounts of pure DNA
Procedure

Isolate DNA
Use restriction enzymes to cut DNA
Ligate fragments into a cloning vector
Transform recombinant DNA into a host to

replicate the DNA and pass copies into

progeny.
 What is genetic engineering or
recombinant DNA technology?
A suite of technologies that enable you to
Isolate and characterise genes
Produce and characterise proteins
Alter the genetic make up of an organism
New genes
Loss of existing genes
 Applications of genetic engineering
Practical applications
 Production of industrially important proteins
 Change the properties of proteins
 Modification of the phenotype of whole organisms
 Diagnosis
 Primary applications in medicine and agriculture
 Others include chemical, paper and detergent industries
 Protein structure and function
 Regulation of gene expression
 Importance of proteins in whole organisms (gene knockouts or null
mutations)

Continue....
Genetically engineered foods
Herbicide resistance
Pesticide resistance
Good for consumer, farmer or biotech. Companies?
Golden rice with increased vitamin A and oil seed rape
with better polyunsaturated fats
Good for consumer?
 Genetic engineering history
Pioneered by Cohen and Boyer 1972-1974 (bacterial
systems)
Southern hybridization 1975
DNA sequencing 1977-1980
Transgenic animals 1980
Polymerase chain reaction 1985
Site-directed mutagenesis 1985
 Where do we start?
If we want to do genetic engineering how do we start?
Isolate the gene of interest
 Select organism containing gene
 Construct a gene library
 Select members of the gene library that contain the gene of interest
Isolate the gene of interest
Lets isolate (clone) a cellulase gene
 Identify organism that contains the gene
 Rumen

 compost

 Soil

 Leaf litter

 Decaying wood
 Gene Cloning
Techniques for gene cloning enable scientists to prepare
multiple identical copies of gene-sized pieces of DNA.
Most methods for cloning pieces of DNA share certain general
features.
For example, a foreign gene is inserted into a bacterial
plasmid and this recombinant DNA molecule is returned to
a bacterial cell.
Every time this cell reproduces, the recombinant plasmid is
replicated as well and passed on to its descendents.
Under suitable conditions, the bacterial clone will make the
protein encoded by the foreign gene.
 ENZYMES USED IN MOLECULAR BIOLOGY

Removes phosphate groups from 5' ends of

Alkaline phosphatase 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
DNA polymerase I template in the 5'-to-3'direction. 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 from
Taq DNA polymerase
a thermostable microbe (Thermus aquaticus)
One basic cloning technique begins with the insertion of
a foreign gene into a bacterial plasmid.
 Restriction Enzymes
In nature, bacteria use restriction
enzymes to cut foreign DNA, such as
from phages or other bacteria.
Most restrictions enzymes are very
specific, recognizing short DNA
nucleotide sequences and cutting at
specific point in these sequences.
 Restriction Endonucleases
The first restriction enzyme to be isolated was that
from E. coli K laboratory strains in 1968 (Meselson
and Yuan, 1968). This enzyme was able to cleave
DNA, but the precise site of DNA cleavage remained
unclear. The enzyme displayed a number of complex
activities that made it difficult to study
The specific DNA sequence is called recognition
sequence
Bacterial restriction–modification systems have two
components – a restriction endonuclease and a DNA
methylase. The restriction enzyme (or restriction
endonuclease) cleaves DNA at specific sequences.
 Restriction Endonucleases
Also called restriction enzymes

1962: “molecular scissors” discovered in in bacteria

E. coli bacteria have an enzymatic immune system that

recognizes and destroys foreign DNA

3,000 enzymes have been identified, around 200 have

unique properties, many are purified and available
commercially
 Restriction Enzymes: Molecular Scissors
Restriction enzymes
(endonuleases) cut DNA at
specific sequences
What kinds of bonds are
broken when restriction
enzymes cut?
Covalent bonds (within a
single strand)
Hydrogen bonds (between
strands) as a result of the Hydrogen
strands coming apart bond
Covalent bond
 Nomenclature
Smith and Nathans (1973) proposed enzyme naming
scheme
three-letter acronym for each enzyme derived from
the source organism
First letter from genus
Next two letters represent species
Additional letter or number represent the strain or
serotypes
For example. the enzyme HindII was isolated from
Haemophilus influenzae serotype d.
Examples of Restriction Enzymes
Enzyme Organism source Recognized
Sequence

EcoRI Escherichia coli 5' GAATTC 3’

3' CTTAAG 5’

TaqI Thermus aquaticus 5' TCGA 3’

3' AGCT 5’
HindIII Haemophilus influenzae 5'AAGCTT 3’
3'TTCGAA 5’
BamHI Bacillus amyloliquefaciens 5' GGATCC 3’
3' CCTAGG 5’
AluI Arthrobacter luteus 5' AGCT 3’
3' TCGA 5’
 Sticky End Cutters

Most restriction enzymes make staggered cuts

Staggered cuts produce single stranded “sticky-ends”
DNA from different sources can be spliced easily
because of sticky-end overhangs.

5’ P
- OH -
3’
HindIII
5’
-P
-
H
3’ O

EcoRI
 Blunt End Cutters
Some restriction enzymes cut DNA at opposite base
They leave blunt ended DNA fragments
These are called blunt end cutters

AluI

HaeIII
Each restriction enzyme cleaves a specific sequence of
bases called a restriction site.
These are often a symmetrical series of four to eight
bases on both strands running in opposite directions.
If the restriction site on one strand is 3’-CTTAAG-5’, the
complementary strand is 5’-GAATTC-3
Restriction enzymes cut covalent phosphodiester bonds of
both strands, often in a staggered way creating single-
stranded ends, sticky ends.
These extensions will form hydrogen-bonded base pairs
with complementary single-stranded stretches on other
DNA molecules cut with the same restriction enzyme
 Restriction Enzymes: EcoRI
• EcoRI (“Echo R one”) is a commonly
used enzyme. It was the first (one)
restriction enzyme isolated from the
“R” strain of E. coli. It demonstrates
the usual type recognition site, a
palindrome (the same on both strands,
reading in opposite directions) EcoRI
leaves a four base, 5’ overhang, sticky
end.
 Cloning DNA
with the
Restriction
Enzyme EcoRI
• A typical DNA cloning experiment requires that the DNA to be cloned (often called
the “insert DNA”) and the vector (often a plasmid) both be cut with the same enzyme
(or with two enzymes which produce compatible ends). The insert DNA and the
vector are then mixed, and DNA ligase is used to join the molecules.
 Ligation of foreign DNA to plasmid vectors
Termini on Requirements for cloning Comments
foreign DNA
Blunt -ended High conc of DNAs and ligase Large no. of non-recombinant
clone
R.E. sites may be eliminated
Tandem copies of foreign DNA
Different protruding Requires purification of plasmid after R.E. sites at junctions are
termini digestion conserved
Low no. of non-recombinants
Foreign DNA is inserted in only
one orientation

Identical protruding Phosphatase treatment of linear R.E. sites at junctions are

termini plasmid vector conserved
Foreign DNA is inserted in either
orientation
Tandem copies of foreign DNA
One goal may be to produce a protein product for use.
A second goal may be to prepare many copies of the
gene itself.
This may enable scientists to determine the gene’s
nucleotide sequence or provide an organism with a new
metabolic capability by transferring a gene from another
organism.
Isolating a cellulase gene

Isolate chromosomal DNA

Fragment DNA
 Mix and ligate

Vector

E. coli

Transform

Gene library
 Cloning Vectors
Plasmid;
Phage;
Cosmid;
Shuttle;
Yeast Artifical Chromosomes (YACs)
Bacterial Artificial Chromosomes (BACs)
Figure 4.2 The first cloning experiment involving a
recombinant DNA assembled in vitro.
Boyer and Cohen cut two plasmids, pSC101 and
RSF1010, with the same restriction
endonuclease, EcoRI. This gave the twolinear
DNAs the same stickyends, which were then
linked in vitro using DNA ligase. The investigators
reintroduced the recombinant DNA into E. coli
cells by transformation and selected clones that
were resistant to both tetracycline and
streptomycin. These clones were therefore
harboring the recombinant plasmid.
 Plasmid Cloning Vectors
Derived from natural plasmids.
Plasmids are circles of dsDNA that include origin
sequences (ori) needed for replication in bacterial
cells.
E.coli plasmid vectors contains
 An ori sequence required for replication in E.coli
 A selectable marker, e.g., antibiotic resistance, to allow selection
of cells harboring the plasmid.
 At least one unique restriction enzyme cleavage site, so that DNA
sequences cut with the RE can be spliced into the plasmid.
Plasmids as Vectors
Using the Ti Plasmid to Transfer Gees to Plants
 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
 DNA FORMS OF A PLASMID
 Uncut plasmid DNA can be
in any of five forms:
▪ nicked
▪ circular
▪ linear covalently closed
▪ supercoiled
▪ circular single-
stranded.
 The exact distances
between the bands of these
different forms is influenced
by:
▪ percentage of agarose
▪ time of electrophoresis
▪ degree of supercoiling
▪ the size of the DNA.
 Linear band = linear size of
plasmid
 PLASMID VECTORS
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
 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
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
 MULTIPLE CLONING SITE

Gene to be cloned can

be introduced into the
cloning vector at one of
the restriction sites
present in the
polylinker
Summary:
The first generations of plasmid cloning vectors were pBR322 and the pUC
plasmids. The former has two antibiotic resistance genes and a variety of unique
restriction sites into which one can introduce foreign DNA. Most of these sites interrupt
one of the antibiotic resistance genes, making screening straightforward. Screening is
even easier with the pUC plasmids. These have an ampicillin resistance gene and a
multiple cloning site that interrupts a partial β-galactosidase gene. One screens for
ampicillin-resistant clones that do not make active β-galactosidase and therefore do
not turn the indicator, X-gal, blue. The multiple cloning site also makes it convenient to
carry out directional cloning into two different restriction sites.
 Figure 4.3 The plasmid pBR322, showing the locations of 11 unique
restriction sites that can be used to insert foreign DNA
The locations of the two antibiotic resistance genes (Ampr =ampicillin
resistance; Tetr =tetracycline resistance) and the origin of replication (ori ) are
also shown. Numbers refer to kilobase pairs (kb) from the EcoRI site.
Figure 4.4 Cloning foreign DNA using the
PstI site of pBR322.
We cut both the plasmid and the insert
(yellow) with PstI, then join them through
these sticky ends with DNA ligase. Next, we
transform bacteria with the recombinant DNA
and screen for tetracycline-resistant,
ampicillin-sensitive cells. The recombinant
plasmid no longer confers ampicillin
resistance because the foreign DNA interrupts
that resistance gene (blue).
Figure 4.5 Screening bacteria by
replica plating.
(a) The replica plating process. We touch a
velvet-covered circular tool to the surface of the
first dish containing colonies of bacteria. Cells
from each of these colonies stick to the velvet and
can be transferred to the replica plate in the same
positions relative to each other. (b) Screening for
inserts in the pBR322 ampicillin resistance gene
by replica plating. The original plate contains
tetracycline, so all colonies containing pBR322
will grow. The replica plate contains ampicillin,
so colonies bearing pBR322 with inserts in the
ampicillin resistance gene will not grow (these
colonies are depicted by dotted circles). The
corresponding colonies from the original plate can
then be picked.
pUC
lacZ’ : coding for the amino terminalportion of the
enzyme β –galactosidease.
Host E.coli strain carry a gene fragment that codes
the carboxyl potion of β –galactosidease;
When X-gal cleaved by β –galactosidease, it
releases galactose plus an indigo dye that stains
the bacterial colony blue.
Figure 4.7 Joining of vector to insert. (a) Mechanism of
DNA ligase.
Step 1: DNA ligase reacts with an AMP donor—either
ATP or NAD(nicotinamide adenine dinucleotide),
depending on the type of ligase. This produces an activated
enzyme (ligase-AMP). Step 2: The activated enzyme
donates a phosphate to the free 5’-phosphate at the nick in
the lower strand of the DNA duplex, creating a high-energy
diphosphate group on one side of the nick. Step 3: With
energy provided by cleavage of the diphosphate, a new
phosphodiester bond is created, sealing the nick in the
DNA. This reaction can also occur in both DNA strands at
once, so two independent DNAs can be joined together by
DNA ligase.
Figure 4.7 Joining of vector to
insert. (b)Alkaline phosphatase
prevents vector re-ligation.
Step 1: We cut the vector(blue, top
left) with BamHI. This produces sticky
ends with 5’-phosphates(red). Step
2: We remove the phosphates with
alkaline phosphatase, making it
impossible for the vector to re-ligate
with itself. Step 3: We also cut the
insert(yellow, upper right) with
BamHI, producing sticky ends with
phosphates that we do not remove.
Step 4: Finally, we ligate the vector
and insert together. The phosphates
on the insert allow two
phosphodiester bonds to form(red),
but leave two unformed bonds, or
nicks, These will be completed once
the DNA is in the transformed
bacterial cell.
 pUC19 (2,686-bp)
High copy number in E.coli, ~100 copies per cell
(makes it easy to purify the plasmid);
Has a selectable marker ampR (indicates the
success of transformation).
A cluster of unique restriction sites, called
polylinker (multiple cloning site makes it easy to
use different RE sites for cloning).
Polylinker is part of lacZ gene (B-galactosidase).
If pUC19 plasmid is put in a lacZ- E. coli, cell will
become lacZ+.
If DNA is cloned into the polylinker, lacZ is
disrupted, no complementation occur.
 Plasmid pUC 19
• The commonly used plasmid pUC19
(“puck 19”) is a small plasmid with the
essential elements for a vector: An origin
of DNA replication A dominant selectable
marked (resistance to an antibiotic,
ampicillin) And a cloning site, usually a
polylinker with recognition sites for
numerous restriction enzymes.
How to detect if DNA is cloned into the polylinker?

X-gal, a chromogenic analog of lactose, truns blue

when B-galactosidase is present, and remains white in
its absence, so blue-white screening can indicate
which colonies contain recombinant plasmids.
 Blue White Screen

• A portion of the lactose utilizing gene (b-galactosidase) is interrupted by the polylinker cloning site.
• Insertion of a DNA fragment prevents expression of the gene.
• Growing the E. coli containing the plasmid on petri plates containing a substrate for the enzyme allows
you to tell those which express the lac-z gene (no insert, blue color) form those colonies that do not
(contain an insert, white color)
Bromo-chloro-indoyl--galactopyranosidase or X-Gal (Clear)

-galactosidase

Bromo-chloro-indoyl (Deep blue insoluble)

galactose
Inserting chromosomal DNA into a vector
Chromosome
Vector GAATTC GAATTC
CTTAAG CTTAAG
GAATTC
CTTAAG Cut with EcoRI and add DNA ligase

Recombinant vector
GAATTC GAATTC
CTTAAG CTTAAG

Ampicillin resistant; -galactosidase negative (White on X-Gal)

LacZ gene codes for -galactosidase

Ampicillin resistance gene

 Wild type vector

GAATTC
CTTAAG

Ampicillin resistant; -galactosidase active (Blue on X-Gal)

LacZ gene codes for -galactosidase

Ampicillin resistance gene

 E. coli sensitive
to ampicillin

Ampicillin resistant; Ampicillin resistant; -galactosidase

-galactosidase active (Blue on X-Gal) negative (White on X-Gal)
Bacteria from ligation plated on ampicillin and X-Gal

Contains
wild type
plasmd

Contains
recombinant
plasmd
 Other plasmid vectors
They contain
different unique restriction enzyme sites
Phage promoters (e.g., T7, T3, SP6) for transcription of
the cloned DNA.
Available for prokaryotic and eukaryotic organisms
Size of the insert is limited; plasmids carrying more
than 5-10 kb are not stable.
 Phage Lambda Vectors
Versions of bacteriophage lambda with sequences for
lysogeny removed, so that only lytic infection is possible.
Lambda replacement vector:
Has a chromosome with a ‘left’ arm and a ‘right’ arm, that
contain all the genes needed for lysis. Between two arms, there
is a disposable segment since it does not contain any lytic cycle
genes. These two regions, the arms and the disposable region is
separated by EcoRI sites. The lambda chromosome central
region is replaced with the insert DNA (~15kb), using RE
digestion and ligation.
 Phage l (lambda) as a Cloning Vector
• Plasmids are limited in the size of DNA that
can be easily introduced into bacteria, about 5-
10 Kb cloned DNA (transformation). By
cloning into a phage, the viral entry system can
be exploited to introduce the DNA into
bacteria. Phage l allows insertion of 15-30 Kb
DNA, with efficient introduction into E. coli.
• Subcloning: transfer of a DNA insert from the
phage clone into a plasmid by having a special
bacterial strain do the work.
Only phages with DNA insert between the two arms can
replicate; Why?
The phage needs both arms to be together for
reproduction and lysis.
Each DNA fragment is cloned by repeated rounds of
infection and lysis. Eventually culture becomes
transparent as all the bacteria has been lysed, and a
population of progeny lambda phages is produced (10 10
to 1011 phages/mL).
 Shuttle Vectors

A cloning vector capable of

replicating in two or more types of
organism (e.g., E.coli and yeast) is
called a shuttle vector. They replicate
autonomously in both hosts or
integrate into them.
Why use shuttle vectors?
They can replicate in two or more host
systems, e.g., replicate in E.coli (it is easy to
do the initial cloning), then be transferred to
yeast. A yeast shuttle vector contains
selectable markers for both systems (ura3 for
yeast; ampR for E. coli); autonomous
replication sites to replicate as a plasmid.
Yeast Artificial Chromosomes
(YACs)
Function as artificial chromosomes in yeast.
Linear structure with a yeast telomere (TEL) at each end.
A yeast centromere sequence (CEN)
A marker gene on each arm that is selectable in yeast (e.g.,
TRP1, URA3).
 Tryptophan and uracil independence in trp1 and ura3 mutant strains,
respectively.
Unique restriction sites for inserting foreign DNA that can be
up to 500kb long. This size of inserts are important in
generating physical maps of genomes.
An origin of replication sequence-ARS (autonomously
replicating sequence)-that allows the vector to replicate in
yeast.
 YACs
Several hundred kb of insert DNA can be
cloned in a YAC. YAC clones are made by:
Generating YAC arms by restriction digest
Ligating with insert fragments up to 500 kb
in length
Transforming into yeast
Selecting for markers (e.g., TRP1 and
URA3).
Why one might want to clone DNA in an
organism other than E.coli.
One can transform yeast as well as
plant and animal cells. This can be
useful in studying cloned eukaryotic
genes in a eukaryotic environment,
commercial production of gene
products (e.g., drugs, antibodies),
developing gene therapy, genetic
engineering.
Yeast Artificial Chromosomes (YACs)
1. YAC vectors function as artificial chromosomes in yeast. Their
features include (Figure 7.8)
a. Linear structure with a yeast telomere (TEL) at each end.
b. A yeast centromere sequence (CEN).
c. A marker gene on each arm that is selectable in yeast. (e.g. TRP1
and URA3)
d. A yeast origin of replication known as autonomous replicating
sequence (ARS).
e. Unique restriction sites for inserting foreign DNA.
2. Several hundred kb of insert DNA can be cloned in a YAC. YAC
clones are made by:
f. generatingYAC arms by restriction digest.
g. Ligating with insert fragments up to 500 kb inlength.
h. Transforming into yeast.
i. Selecting for markers (e.g. TRP1 and URA3)
Yeast Artificial Chromosome Chromosomal DNA
centromere
origin
of replication sup4 gene

Ura3 gene
SnaBI site Telomeres
Trp1
gene
BamHI sites

Cut with SnaBI

0.1-1 Mb
Mix and add
Cut with
SnaBI and DNA ligase
BamHI

Telomere Trp1 Ori Cent sup4’ chromosomal DNA sup4’ Ura3 Telomere

Transform recombinant YACs into mutant yeast

that lack Ura3 and Trp1 genes (I.e. can’t make tryptophan
and uracil) so the amino acid and nucleotide must
be added for yeast to grow
Essential components of YAC vectors
•Centromers (CEN), telomeres (TEL) and
autonomous replicating sequence (ARS) for
proliferation in the host cell.
•ampr for selective amplification and markers such
as TRP1 and URA3 for identifying cells containing
the YAC vector.
•Recognition sites of restriction enzymes (e.g.,
EcoRI and BamHI)
 Transformed yeast plated on media lacking tryptophan and uracil

Colony contains
Colony contains
wild type YAC
recombinant YAC

Plate yeast on medium lacking uracil and

tryptophan. Yeast colonies that grow contain YAC
and those that are pink contain recombinant YAC
as this indicates inactivation of Sup 4
Telomere Centromere Telomere

Origins of
replication
Accepts up to 1 Mb of chromosomal DNA

Yeast Artificial Chromosome (YAC)

Accepts up to 1 Mb of DNA
centromere
origin
of replication sup4 gene

Ura3 gene
SnaBI site
Trp1
gene
BamHI sites Telomeres
 M13 , a single stranded filamentous phage

 Phage DNA is packaged in the core of a helical particle

 The length of particle is dependant on length of DNA
 In all M13 preps the following occur
 polyphage- more than one genome length
 minphage- 0.2-0.5 genome length
 maxiphage- genetically defective but more than one genome length
 Molecular biologists use this to create cloning vectors
 Can insert long stretches of DNA into non essential regions
 No sharp cut off in length that can be packaged
 Some decrease in efficiency of packaging with increasing length
 10% longer not affected, 50% longer replicate more slowly
How M13 infects and reproduces
 infects through pili
 Protein coat is stripped and ss DNA is converted to double stranded
replicative form
 DNA relicated by “rolling circle method”
 New particles assembled
 200 particles per infected cell per generation
 M13 released without lysis
 No lysis on bacterial lawn, generally do in liquid culture.
Uses

 Cloning
 Ss DNA for probes, sequencing
 Phage display technology, M13 will produce foreign protein on surface
as part of its protein coat, can use to generate specific antibodies
 Figure 4.10 Obtaining single-stranded
DNA by cloning in M13 phage.
Foreign DNA (red), cut with HindIII, is
inserted into the HindIII site of the
double-stranded phage DNA. The
resulting recombinant DNA is used to
transform E.coli cells, whereupon the
DNA replicates by a rolling circle
mechanism, producing many single-
stranded product DNAs. The product
DNAs are called positive (+) strands, by
convention. The template DNA is
therefore the negative (-) strand.
 Phagemides
Single-stranded;
Both phage and plasmid
characteristics;
Help phage
Two RNA polymerase promoters (T7and
T3)
 Summary
Two kinds of phages have been especially popular as cloning vectors. The
first of these is λ, from which certain nonessential genes have been removed
to make room for inserts. Some of these engineered phages can accommodate
inserts up to 20 kb, which makes them useful for building genomic libraries,
in which it is important to have large pieces of genomic DNA in each clone.
Cosmids can accept even larger inserts—up to 50 kb—making them a
favorite choice for genomic libraries. The second major class of phage vector
is composed of the M13 phages. These vector have the convenience of a
multiple cloning site and the further advantage of producing single-stranded
recombinant DNA, which can be used for DNA sequencing and for site-direct
mutagenesis. Plasmids called phagemids have also been engineered to
produce single-stranded DNA in the presence of helper phages.
 Recombinant DNA libraries
Genomic library
Chromosome library
cDNA library
 Genomic libraries
Are constructed by digesting genomic DNA
Complete digestion
Mechanical shearing
Partial digestion
 Partial digestion
They are selected in a certain size range by density
gradient centrifugation or agarose gel
electrophoresis
DNA fragments from sticky ends can be cloned
directly.
Genomic sequences are not equally represented in
the library
Regions of DNA with relevant restriction sites very close
together or far apart are removed at the selection stage
Some regions prevent vector replication so eliminated.
Partial Digest for Producing Clonable Fragments

Enzymes with compatible

Sticky ends are used.
How many clones are needed to contain all sequences
in genome?
Depends on:
Size of the genome being cloned
The average size of the DNA fragments inserted into the
vector.
 How many clones are needed to contain all
sequences in genome?
The probability of having at least one copy of any DNA
sequence in the genomic library is:
N=ln(l-P)/ln(1-f),
Where N = the necessary number of recombinant DNA
molecules;
P = the probability desired;
F = average size of the fragments divided by the genome size
Ln = natural algorithm
Within the human genome (3 x 109 bp), how many 40-kb
pieces would you have to clone into a library if you
wanted to be 90% certain of including a particular
sequence?
P = 0.90; f=40,000/(3x109)
N=ln(l-P)/ln(1-f) = 172,693 fragments
 cDNA libraries
cDNA drives from mature mRNA, no introns.
polyA tail at the 3’ is useful for:
Isolating mRNA from cell lysates
Priming the synthesis of cDNA providing a known 5’
sequence
Linkers for Cloning DNA
• Any DNA fragment can have a
specific restriction site added to
the ends by ligating on a
“linker”.
• Linkers are small, synthetic
(made in the lab, or ordered
from a company) DNA
fragments which contain the
recognition sequence for one or
more restriction enzymes.
• After ligating on linkers, the
DNA is cut with the
appropriate restriction enzyme
to produce ends for cloning.
Random Primed DNA Synthesis for Making a “Probe
• One common technique for making a
probe to detect a specific DNA sequence
is called “random primed DNA
synthesis”.
• Short (8 bases) primers of random
sequence are annealed to the heat
denatured DNA (of the sequence for the
probe), and DNA polymerase is used to
synthesize copies of the DNA with one of
the nucleotides incorporated carrying a
detectable label (such as radioactive
phosphate).
Recognition sequence Recognition frequency

4 bp 44 =254 bp
6 bp 46=4096 bp
8 bp 48=65000bp Rere cutters
The main advantage of restriction enzyme is there
ability to cut a DNA reproducibly in the same place;
this is the basis of many techniques used to analyze
genes.

Many restriction enzymes make staggered cut in the

two DNA strands, leaving a sticky ends, that can base-
pair together briefly.

Enzymes that recognize identical sequences are called

isoschizomers.
 Expression Vectors
 Expression vectors with strong promoters
 Inducible Expression Vectors
 Expression vectors produce fusion proteins
 Eukaryotic expression vectors
Scheme for using phage  DNA as a cloning vector
 Cloning DNA into vectors
cos
Left arm Central Right arm Chromosomal DNA

Cut chromosome with

Cut out 3 regions same enzyme as lambda
and purify arms

Mix DNA and add

DNA ligase

DNA
will form
concatamers
Will not package Recombinant lambda will
package. Why?
DNA packaging is size dependent

Endonuclease A
cleaves cos sequence

DNA too small

to package <18 kb
cos
DNA packages
18-25 kb

DNA too large to

package >25 kb
 Cosmid vector
Cos

GGATCC GGATCC
CCTAGG CCTAGG
Ampr
Cut with BamHI

Cut with BamHI

Origin GATCC G
G CCTAG
BamHI
GATCC G
G CCTAG Mix DNA at very
high concentration
and add DNA ligase
GGATCC GGATCC GGATCC GGATCC
GGATCC GGATCC GGTACC GGATCC

Packaged in vitro
 Phage λ Cloning Vectors
1. There are versions of bacteriophage λ (lamda) with sequences for
lysogeny removed, so that only lytic infection is possible.
2. Restriction sites in the λ DNA separate required bacteriophage
genes in the chromosome arms from the nonessential sequences in
the central region. The λ chromosome central region is replaced
with insert DNA, using restriction digestion and DNA ligation.
3. When ligated DNA is mixed with phage λ proteins, phage heads
assemble and DNA is packaged, forming virus paritcles.
4. Only phage with both arms of phage λ chromosome and a
properly sized (37-52kb) central insert sequence are able to
replicate by infected E. coli. Progeny phages include insert DNA
in their chromosomes, so do their progeny in the following rounds
of infection, providing quantities of the insert DNA.
5. Many versions of phage cloning vectors are available, with
features including and expanded array of restriction sites, systems
for expressing cloned genes and for creating plasmid subclones of
the insert DNA.
Cosmid cloning vector
 Shuttle Vectors
1. A cloning vector capable of replicating in two or more
types of organism (e.g., E. coli and yeast) is called a
shuttle vector. Shuttle vectors may replicate
autonomously in both hosts, or integrate into the host
genome
 Cosmid vectors
Problem with vectors
is that you can’t Cosmid vector
Cos
transform large pieces
of DNA into E. coli
Cosmid
Similar to plasmid Ampr
 Ampicillin resistance
gene
 5 kb in size
 Unique BamHI site
Origin
 Cos sequence
BamHI
Lambda particle
injects cosmid in E. coli.
E. coli is viable as
no lambda genes in How do you select
cosmid so it acts as for E. coli cells containing
a normal plasmid cosmids?

They are resistant

to ampicillin
Bacterial Artificial Chromosomes (BACs)

1. BACs are used for cloning fragments up to about 200

kb in E .coli. BAC vectors contain:
a. the ori of an E. coli plasmid called the F factor.
b. A multiple cloning sites.
c. A selectable marker.
d. Other features, “bells and whistles.”
2. BAC can be handles like regular bacterial plasmids,
but the F factor ori keeps copy number at one BAC
molecule per cell.
 BAC vectors
Bacterial artificial chromosomes are based on the F factor
of E. coli and can be used to clone up to 350 kb of genomic
DNA in a conveniently handled E. coli host. They are a morre
stable and easier to use alternative to YAC.
 T-DNA has almost identical repeats of 25 bp at each end in the Ti plasmid. The right repeat is
necessary for transfer and integration to a plant genome. T-DNA that is integrated in a plant
genome has a precise junction that retains 1-2 bp of the right repeat, but the left junction varies
and may be up to 100 bp short of the left repeat.
Figure 4.24 Crown gall tumors. (a) Formation of a crown gall.
1. Agrobacterium cells enter a wound in the plant, usually at the crown, or the junction of root an
stem. 2. The Agrobacterium contains a Ti plasmid in addition to the much larger bacterial
chromosome. The Ti plasmid has a segment (the T-DNA, red) that promotes tumor formation in
infected plants. 3. The bacterium contributes its Ti plasmid to the plant cell, and the T-DNA from
the Ti plasmid into grates into the plant’s chromosomal DNA. 4. The genes in the T-DNA direct
the formation of a crown gall, which nourishes the invading bacteria.
 Nopaline and octopine Ti plasmids carry a variety of
genes, including T-regions that have overlapping functions
Figure 4.24 Crown gall tumors. (b) Photograph of a crown gall tumor
genetated by cutting off the top of a tobacco plant and inoculating
with Agrobacterium.
This crown gall tumor is a teratoma, which generates normal as well as tumorous
tissues springing from the tumor.
Figure 4.25 Using a T-DNA plasmid to
introduce a gene into tobacco plant.
(a) A plasmid is formed with a foreign
gene (red) under the control of the mannopine
synthetase promoter (blue). This plasmid is
used to transform Agrobacterium cells.
(b) The transformed bacterial cells divide
repeatedly. (c) A disk of tobacco leaf tissue is
removed and incubated in nutrient medium,
along with the transformed Agrobacterium
cells. These cells infect the tobacco tissue,
transferring the plasmid bearing the cloned
foreign gene. (d) The disk of tobacco tissue
sends out roots into the surrounding medium.
(e) One of these roots is transplanted to
another kind of medium, where it forms a
shoot. This plantlet grows into a transgenic
tobacco plant that can be tested for expression
of the transplanted gene.
 Summary:
Molecular biologists can clone hundreds of thousands of
base pairs of DNA at a time in yeast artificial chromosomes
(YACs). If they wish to transfer cloned genes to plants,
creating transgenic organisms with altered characteristics,
they use a plant vector such as the Ti plasmid.
Size of gene library
N = ln(1-P)
ln (1-A/B)

N = Number of clones
P = 95 % probability of finding gene
A = Average size of DNA fragments
B = Total size of genome

E. coli has genome of 4,800,000 nucleotides

Average size of insert is 10,000 nucleotides
Number of clones for 95 % probability is 1700
 Size of gene library
If genome is large e.g. human genome (3 x 109)
then number of clones to make library becomes
unrealistic (1058000) if using a plasmid vector
(accepts only 10 kb as larger DNA can’t be
transformed)
Therefore need to use vectors that can accept larger
pieces of DNA
I.e. if each vector contains a large piece of DNA you
don’t need so many clones to make a gene library
 Lambda Vector
Lambda vector
Infects E. coli replicates and then viruses
released
End of genome are 12 bp sequences known as
cos sequences.
Cos sequences play an important role in
packaging viral DNA into capsids (head of the
virus)
 Lambda infects E. coli

DNA injected
into E. coli

DNA replication generating

concatamers
Lambda DNA is linear in virus
Cos sequence is 12 nucleotides and single stranded
The two cos sequences are complementary

cos sequences hybridise in E.coli

to form circular genome

Replicates by rolling ciricle in E. coli

to produce concatemers
cos lambda DNA cos etc etc
More information on vectors
Lambda vector
Libraries contain larger inserts than plasmids (20-
25 kb)
Naked Lambda DNA can’t be transformed into E.
coli
Lambda DNA can be packaged into a virus
Virus then infected into E. coli
 What vectors for what libraries?
Vector Insert What libraries
size
Plasmid <10 kb Bacteria

Lambda phage 18-25 kb Yeast

Cosmid 34-45 kb Intermediate
eukaryotes
YAC etc 0.1 – 1 Mb Higher Eukaryotes
Human library requires >1,000,000 plasmid clones

Human library requires 14000 YAC clones

Screening bacterial gene library for a specific gene

Assume bacterial genes will express in Escherichia

coli
Screen for gene by looking for protein that
Changes phenotype of E. coli
 e.g. confers ability to degrade a polysaccharide (cellulase,
xylanase etc) confers green fluorescence (green fluorescent
protein)
Changes phenotype of an E. coli mutant. Known as
complementation
Screen for protein directly using an antibody
 Complementation
Isolate gene from a bacterium that E. coli
contains
e.g. Isolate B. subtilis gene coding 1st enzyme in
leucine biosynthsis pathway
Assume bacteria have same pathway for leucine
synthesis
Step 1: Isolate a mutant of E. coli unable to
synthesise leucine
I.e. gene for 1st enzyme non-functional thus
enzyme not produced
 Complementation
(generating a leucine auxotroph)

Plate out on
media containing
Add mutagen leucine

E. coli culture Mutated E.coli culture

Transfer
Colony 4 requires colonies
leucine for growth to media
(leucine auxotroph) lacking leucine
 Complementation
Leucine auxotrophs isolated require leucine for
growth
Defect in gene coding for leucine synthesis
Which gene in pathway mutated?
Assay leucine auxotrophs for mutant lacking enzyme
one
 Complementation
Construct a gene library of Bacillus subtilis
chromosomal DNA
Digest chromosome
Insert into E. coli vector to make a library of
recombinant vectors
Transform these recombinant vectors into the E.
coli leucine auxotroph lacking enzyme 1
Plate out library on media lacking leucine
 Complementation
E. coli colony that grows contains recombinant
plasmid with Bacillus leucine biosynthetic gene that
codes for 1st enzyme in leucine synthesis
The recombinant plasmid has overcome or
complemented the E. coli mutation
 Screening
Bacterial gene library
Depends on protein expression
 Phenotype change e.g. cellulase gene
 Complementation of E. coli mutant
 Detection of gene product using antibody

Eukaryotic gene library

No protein expression expected
Nucleic acid hybridisation
Probe
 Homologous gene from other organism
 Oligonucleotide probe based on protein sequence
 Antibody screening
Some bacterial genes code for proteins that don’t
change phenotype of E. coli wild type or mutant e.g.
asparaginase from Erwinia
Erwinia asparaginase converts asparagine to
aspartic acid
Used as a major ingredient of chemotherapy of
childhood acute leukaemia
IDENTIFICATION OF POSITIVE CLONES
One of the first steps is to identify clones carrying the
recombinant plasmid, with the desired DNA insert.
This can be done by 'picking' clones - choosing individual
bacterial colonies in order to isolate the plasmid DNA from
each of them.
Single bacterial colonies are grown in culture broth containing
the selection antibiotic in order to maintain the plasmid.
The plasmid DNA is extracted by the standard minipreparation
technique and then analysed by restriction digest.
After digesting the DNA, different sized fragments are
separated by agarose gel electrophoresis and the sizes
determined by comparison with known DNA molecular weight
marke
Example of a yeast artificial chromosome (YAC) cloning vector
 DNA Sequencing
Animation: Dideoxy DNA Sequencing
1. Once cloned, DNA fragments may be sequenced,
allowing identification of gene and regulatory
sequences, and comparison with homologous genes
from different organisms.
2. Dideoxy sequencing (Sanger, 1970s) is based on DNA
polymerase extending short primers, using either
linear or circular DNA as a template. In dideoxy
sequencing (Figure 7.19):
a. DNA is heat denatured, and a short oligonucleotide primer
(designed so 3’ end is near DNA sequence of interest) anneals
to one strand and serves as primer.
b. A reaction mix is set up with label on either the primer or
dNTP(s). The mix includes:
i. ssDNA template to be sequenced.
ii. Primer (anneals to template).
iii. DNA polymerase.
iv. The four deoxynucleotide (dNTP) precursors.
Dideoxy DNA sequencing of a theoretical DNA fragment
c. The reaction mix is divided into four tubes, and to each is added a
small amount of a different dideoxynucleotide, so that one tube
receives ddCTP, another ddTTP, etc. (Figure 7.20).
d. Dideoxynucleotides lacks 3’-OH, having only 3’-H, and so are
unable to bond with the 5’-phosphate of another nucleotide. A
phosphodiester linkage cannot form, and the chain terminates at
the site of insertion of the ddNTP.
e. The specific ddNTP in a reaction competes with its corresponding
NTP for incorporation into the growing DNA chain.
f. The four reaction mixes, one for each ddNTP, are typically run in
adjacent lanes on a polyacrylamide gel. The label on the DNA
bands reveals their location, and therefore their sizes.
g. DNA sequence is determined by reading the sequencing ladder
from bottom to top to give the sequence of the newly synthesized
strand from 5’3’ (Figure 7.21).
A dideoxynucleotide (ddNTP) DNA precursor
3. Automation based on the dideoxy method enables
rapid DNA sequencing. In the automated process:
a.Only one reaction mix is needed, containing all four
dideoxynucleotides, each tagged with a different color.
b.DNA fragments generated are separated by
electrophoresis in a single lane.
c.The gel is scanned by a laser device that determines
which fluorescent label is present at each nucleotide
position in the sequence, and sends the information to a
computer.