BB211: Cell and Molecular Biology

Dr Eve Lutz
Department of Bioscience
Recombinant DNA technology:
Lecture 3
Recombinant DNA: Plasmids,
cloning
Background reading:
Reference for this lecture please read the following:
Chapter 16
Klug, WS & Cummings, MR Essentials of Genetics, 4th ed.
What is DNA cloning?
DNA cloning is the isolation of a fragment or fragments of DNA
from an organism and placing in a VECTOR that replicates
independently of chromosomal DNA. The RECOMBINANT DNA is
propagated in a host organism; the resulting CLONES are a set of
genetically identical organisms which contain the recombinant
DNA.

Why is DNA cloning important?

DNA cloning is involved in a number of applications (GENETIC
ENGINEERING). Many techniques for DNA isolation and
manipulation have been worked out and are now routine in
scientific laboratories. These techniques are now important tools
that every scientist must know about.

Three main purposes for cloning DNA

1) DNA sequencing
Determining the sequence of the bases in the DNA can tell us
 about which proteins or RNAs are encoded and their sequences,
which sequences control their expression (GENE PROMOTERS
and other control sequences), as well as any possible mutations
which might alter their function. Having access to the complete
DNA sequence of an organism can help us decipher the biology of
that organism.

2) Protein production
Isolating the gene which encodes a desired protein (haemoglobin,
interferon) may allow that gene to be over-expressed so that the
protein can be produced in bulk for study or use

3) Engineering animals/plants/proteins
The ability to alter the properties of proteins as well as create
genetically modified animals and plants (TRANSGENICS) has lead
to their use for research and for therapeutic and commercial
purposes. The technology may lead to the development of new
therapies for the treatment of disease (GENE THERAPY).

Cloning and Expression Vectors

Isolated DNA is cloned into VECTORS for long term storage,
propagation of the DNA and for production of protein from
gene(s) encoded in the DNA

What are cloning vectors?

Cloning vectors are extra-chromosomal 'replicons' of DNA which can be
isolated and can replicate independently of the chromosome. Vectors
usually contain a selectable marker - a gene that allows selection of
cells carrying the vector e.g. by conferring resistance to a toxin. DNA
of interest can be inserted into the vector and replicated in host cells,
usually one which has been well characterized.

Commonly used vector systems

• Bacterial plasmids
• Bacteriophages
 • Cosmids
• Yeast artificial chromosomes (YACs)
• Ti plasmid (plants)
• Eukaryotic viruses such as baculovirus (insect cells), SV40
virus and retroviruses.

Plasmids are the most commonly used vector system. Several

types are available for cloning of foreign DNA in the host
organism Escherichia coli. Many E. coli plasmids allow the
expression of proteins encoded by the cloned DNA

Bacteriophage are another common vector system used for

cloning DNA. These are viruses which 'infect' E. coli. The M13
bacteriophage is a single-stranded DNA virus which replicates in
E. coli in a double-stranded form that can be manipulated like a
plasmid. It can be used to produce single-stranded DNA copies
which are useful for DNA sequencing.

Bacteriophage λ is another bacteriophage which is commonly used

to make DNA libraries. It allows the cloning of larger fragments
of DNA than can be incorporated into plasmids.

Strategy for cloning DNA into a plasmid (or

other cloning) vector

SUBCLONING
• cut DNA of interest with the appropriate restriction
endonuclease(s)
• separate fragments by gel electrophoresis
• purify target fragment from gel
• ligate fragment with a plasmid cut with the same restriction
endonuclease(s) - ligation is performed using the enzyme T4
DNA ligase, ATP and Mg2+ ions

Subcloning an EcoRI fragment into a plasmid

cloning vector
Good efficiency of ligation of foreign DNA into a vector can be
achieved if both the vector and the insert DNA are cut with 2
different restriction enzymes which leave single stranded ends
(cohesive ends). The DNA is ligated in only one direction, and
there is only a low background of non-recombinant plasmids.
If only one restriction enzyme is used to cut the vector and
insert, then efficiency of ligation is lower, DNA can be inserted in
two directions and tandem copies of inserts may be found. To
avoid high background of non-recombinants, alkaline phosphatase
is used to remove 5' phosphate groups from the cut vector to
prevent self-ligation.
If vector and insert DNA are cut with an enzyme which leaves
blunt DNA ends, the background of non-recombinant plasmids can
be high and the best way around the problem is to use high
concentrations of both DNAs and of the DNA ligase enzyme.

Transformation is the process by which plasmids (or other DNA)

can be introduced into a cell. For E. coli transformation with
plasmids is quite straightforward, plasmids can be introduced by
electroporation or by incubation in the presence of divalent
cations (usually Ca2+) and a brief heat shock (42°C) which induces
the E. coli cells to take up the foreign DNA

There are different methods to select for transformed cells. For

instance, transformants can be selected as antibiotic-resistant
colonies on agar plates containing antibiotic.

For E. coli transformed with plasmids, colonies grown on

antibiotic-containing plates should all carry plasmids. However,
this does not guarantee that the plasmid contains an insert. It is
possible that the vector has re-ligated and not incorporated an
insert.
 A means to determine which clones contain plasmids with inserts
is to use a positive control method such as insertional
inactivation. This provides a clear way of recognising
recombinants from those carrying re-ligated vector.

1. two antibiotic selection and replica plating

2. colour selection: blue/white selection using the lacz gene

Analysis of 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 markers.

Restriction enzymes are a useful tool for

analysing Recombinant DNA
• Checking the size of the insert
• Checking the orientation of the insert
• Determining pattern of restriction sites within insert DNA

Sometimes it is important to determine the orientation of the

DNA insert in relation to the vector sequence. This can be done
simply by restriction digest using enzyme(s) which cut the vector
sequence near to the insert and cut within the insert sequence
(asymmetrically).
 Figure of agarose gel stained with ethidium bromide and
visualised with uv light

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