CHAPTER 4

Cloning, Expression
and Analysis of Genes
and Their Products
4.1 Concept of
molecular cloning

AN OVERVIEW
Molecular cloning takes advantage of the fact that the
chemical structure of DNA is fundamentally the same in
all living organisms.
Therefore, if any segment of DNA from any organism
is inserted into a DNA segment containing the
molecular sequences required for DNA replication, and
the resulting recombinant DNA is introduced into the
organism from which the replication sequences were
obtained.
Then the foreign DNA will be replicated along with
the host cell's DNA in the transgenic organism.

THE CONCEPT
Molecular cloning is a set of experimental methods
in molecular biology that are used to assemble
recombinant DNA molecules and to direct their
replication within host organisms
or Molecular cloning is a basic technique used in a
molecular biology labs.
For eg : a techniques for isolating the luciferase
gene (luc) from DNA using restriction digestion and
cloning it into the multiple cloning region of a vector.
The cloned luc gene is then expressed in E. coli

CLONING
The use of the word cloning refers to the fact that the
method involves the replication of a single DNA
molecule starting from a single living cell to generate a
large population of cells containing identical DNA
molecules
generally uses DNA sequences from two different
organisms
(i)
the species that is the source of the DNA to be
cloned and
(ii) the species that will serve as the living host for
replication of the recombinant DNA.

WRITE ABOUT CLONING

C O N V E N T I O N A L LY
In a conventional molecular cloning experiment, the DNA to be cloned is obtained
from an organism of interest, then treated with enzymes in the test tube to
generate smaller DNA fragments. Subsequently, these fragments are then
combined with vector DNA to generate recombinant DNA molecules. The
recombinant DNA is then introduced into a host organism (typically an easy-togrow, benign, laboratory strain of E. coli bacteria). This will generate a population
of organisms in which recombinant DNA molecules are replicated along with the
host DNA. Because they contain foreign DNA fragments, these are transgenic or
genetically modified microorganisms (GMO).[ This process takes advantage of the
fact that a single bacterial cell can be induced to take up and replicate a single
recombinant DNA molecule. This single cell can then be expanded exponentially to
generate a large amount of bacteria, each of which contain copies of the original
recombinant molecule. Thus, both the resulting bacterial population, and the
recombinant DNA molecule, are commonly referred to as "clones". Strictly
speaking, recombinant DNA refers to DNA molecules, while molecular cloning
refers to the experimental methods used to assemble them.

STEPS IN MOLECULA
R CLONING
1. Choice of host organism and cloning vector
2. Preparation of vector DNA
3. Preparation of DNA to be cloned
4. Creation of recombinant DNA with DNA ligase
5. Introduction of recombinant DNA into host orga
nism
6. Selection of organisms containing vector sequen
ces
7. Screening for clones with desired DNA inserts a
nd biological properties

FACTS TO REMEMBER
Molecular
cloning
is
similar
to
polymerase chain reaction (PCR) in that
it permits the replication of DNA
sequence. The fundamental difference
between the two methods is that
molecular cloning involves replication
of the DNA in a living microorganism,
while PCR replicates DNA in an in vitro
solution, free of living cells.

STEP 1 : CHOICE OF HOST ORGANISM AND

CLONING VECTOR

The
great
majority
of
molecular
cloning
experiments begin with a laboratory strain of the
bacterium E. coli and a plasmid cloning vector .
E. coli and plasmid vectors are in common use
because
they
are
technically
sophisticated,
versatile, widely available, and offer rapid growth of
recombinant organisms with minimal equipment.
If the DNA to be cloned is exceptionally large
(hundreds of thousands to millions of base pairs),
then
a
bacterial artificial chromosome
or
yeast artificial chromosome vector is often chosen.

STEP 1 : CHOICE OF HOST

ORGANISM AND CLONING VECTOR
Specialized applications may call for specialized host-vector
systems.
For example, if the experimental lists wish to harvest a
particular protein from the recombinant organism, then an
expression vector is chosen that contains appropriate signals
for transcription and translation in the desired host organism.
Alternatively, if replication of the DNA in different species is
desired (for example transfer of DNA from bacteria to plants),
then a multiple host range vector (also termed shuttle vector)
may be selected. In practice, however, specialized molecular
cloning experiments usually begin with cloning into a
bacterial plasmid, followed by subcloning into a specialized
vector.

STEP 1 : CHOICE OF HOST

ORGANISM AND CLONING VECTOR
Whatever combination of host and vector are used, the
vector almost always contains four DNA segments that are
critically important to its function and experimental utility
(1) an origin of DNA replication is necessary for the vector
(and recombinant sequences linked to it) to replicate inside
the host organism
(2) one or more unique restriction endonuclease
recognition sites that serves as sites where foreign DNA may
be introduced
(3) a selectable genetic marker gene that can be used to
enable the survival of cells that have taken up vector
sequences, and (4) an additional gene that can be used for
screening which cells contain foreign DNA