INTRODUCTION

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.[1] The
use of the word cloning refers to the fact that the method involves the replication of one molecule
to produce a population of cells with identical DNA molecules.

Molecular cloning generally uses DNA sequences from two different organisms: the species that
is the source of the DNA to be cloned, and the species that will serve as the living host for
replication of the recombinant DNA. Molecular cloning methods are central to many
contemporary areas of modern biology and medicin

Early Genetic Engineering Experiment Once genes could be recombined into cloning vectors,
biologists sought to clone specific genes from various organisms. However, it was evident that
cloning eukaryotic DNA into bacterial hosts would be problematic. This is because eukaryotic pre-
mRNA must be processed and bacteria lack the molecular machinery to perform this task.

In 1970 Howard Temin and David Baltimore independently discovered the enzyme that solved this
dilemma. They isolated the enzyme reverse transcriptase (RT) from retroviruses. These viruses have
an RNA genome copied into DNA before replication. The mechanism by which reverse transcriptase
accomplishes this is outlined in figure 17.5. Processed mRNA can be used as a template for
complementary DNA (eDNA) synthesis in vitro. The resulting eDNA can then be cloned without
needing RNA processing.

Gene Manipulations

The ability to manipulate DNA with precision in a test tube or an organism, known as recombinant
DNA technology has had a dramatic impact on all aspects of cell and molecular biology, allowing us
to routinely study cells and their macromolecules in ways that were unimaginable even twenty years
ago. Central to the technology are the following manipulations:

1. Cleavage of DNA at specific sites by restriction nucleases greatly facilitates the isolation and
manipulation of individual pieces of a genome.
2. DNA ligation makes it possible to seamlessly join together DNA molecules from widely
different sources.
3. DNA cloning (through the use of either cloning vectors or the polymerase chain reaction) in
which a portion of a genome (often an individual gene) is “purified” away from the
remainder of the genome by repeatedly copying it to generate many billions of identical
molecules.
4. Nucleic acid hybridization, which makes it possible to identify any specific sequence of DNA
or RNA with great accuracy and sensitivity based on its ability to selectively bind a
complementary nucleic acid sequence.
5. DNA synthesis makes it possible to chemically synthesize DNA molecules with any sequence
of nucleotides, whether or not the sequence occurs in nature.
6. Rapid determination of the sequence of nucleotides of any DNA or RNA molecule
Restriction Enzymes

all DNA molecules consist of an approximately equal mixture of the same four nucleotides, they
cannot be readily separated, as proteins can based on their different charges and biochemical
properties. The solution to this problem began to emerge with the discovery of restriction nucleases.
These enzymes, which are purified from bacteria, cut the DNA double helix at specific sites defined
by the local nucleotide sequence, thereby cleaving a long, double-stranded DNA molecule into
fragments of strictly defined sizes. They are of high specificity and produce either blunt end or sticky
ends.

(1) Choice of host organism and cloning vector-

Although a very large number of host organisms and molecular cloning vectors are in use, the
great majority of molecular cloning experiments begin with a laboratory strain of the
bacterium E. coli (Escherichia coli) and a plasmid cloning vector. 

Specialized applications may call for specialized host-vector systems. For example, if the
experimentalists 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

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:[3]

 DNA replication origin is necessary for the vector (and its linked recombinant
sequences) to replicate inside the host organism
 one or more unique restriction endonuclease recognition sites to serves as sites
where foreign DNA may be introduced
 a selectable genetic marker gene that can be used to enable the survival of cells that
have taken up vector sequences
 a tag gene that can be used to screen for cells containing the foreign DNA

(2) Preparation of vector DNA-

The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where
foreign DNA will be inserted. The restriction enzyme is chosen to generate a configuration at the
cleavage site that is compatible with the ends of the foreign DNA (see DNA end). Typically, this
is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme, for
example EcoRI. Most modern vectors contain a variety of convenient cleavage sites that are
unique within the vector molecule (so that the vector can only be cleaved at a single site) and
are located within a gene (frequently beta-galactosidase) whose inactivation can be used to
distinguish recombinant from non-recombinant organisms

Preparation of DNA to be cloned

For cloning of genomic DNA, the DNA to be cloned is extracted from the organism of interest.
Virtually any tissue source can be used as long as the DNA is not extensively degraded. The
DNA is then purified using simple methods to remove contaminating proteins (PCR) methods
are often used for amplification of specific DNA sequences prior to molecular cloning.
DNA for cloning experiments may also be obtained from RNA using reverse transcriptase
(complementary DNA or cDNA cloning), or in the form of synthetic DNA (artificial gene
synthesis). cDNA cloning is usually used to obtain clones representative of the mRNA population
of the cells of interest, while synthetic DNA is used to obtain any precise sequence defined by
the designer.
The purified DNA is then treated with a restriction enzyme to generate fragments with ends
capable of being linked to those of the vector.

(3) Creation of recombinant DNA

DNA prepared from the vector and foreign source are simply mixed together at appropriate
concentrations and exposed to an enzyme (DNA ligase) that covalently links the ends together.
The resulting DNA mixture containing randomly joined ends is then ready for introduction into the
host organism.
The desired products vector DNA covalently linked to foreign DNA will be present, but other
sequences) are also usually present. This complex mixture is sorted out in subsequent steps of
the cloning process, after the DNA mixture is introduced into cells.

5) Introduction of recombinant DNA into the host organism

The DNA mixture, previously manipulated in vitro, is moved back into a living cell. The methods
used to get DNA into cells are varied, and the name applied to this step in the molecular cloning
process will often depend upon the experimental method that is chosen [11]
When microorganisms are able to take up and replicate DNA from their local environment, the
process is termed transformation, and cells that are in a physiological state such that they can
take up DNA are said to be competent.[15] In mammalian cell culture, the analogous process of
introducing DNA into cells is commonly termed transfection. Both transformation and transfection
usually require preparation of the cells through a special growth regime and chemical treatment
process that will vary with the specific species and cell types that are used.
Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane
(and cell wall, if present).[16] In contrast, transduction involves the packaging of DNA into virus-
derived particles, and using these virus-like particles to introduce the encapsulated DNA into the
cell through a process resembling viral infection. Although electroporation and transduction are
highly specialized methods, they may be the most efficient methods to move DNA into cells.

(4) Selection of organisms containing recombinant

DNA
Whichever method is used, the introduction of recombinant DNA into the chosen host
organism is usually a low efficiency process; that is, only a small fraction of the cells will
actually take up DNA. Experimental scientists deal with this issue through a step of artificial
genetic selection, in which cells that have not taken up DNA are selectively killed, and only
those cells that can actively replicate DNA containing the selectable marker gene encoded
by the vector are able to survive
(5) Screening for clones with desired DNA inserts
and biological properties.
Modern bacterial cloning vectors use the blue-white screening system to distinguish colonies
(clones) of transgenic cells from those that contain the parental In these vectors, foreign DNA is
inserted into a sequence that encodes an essential part of beta-galactosidase, an enzyme
whose activity results in formation of a blue-colored colony on the culture medium that is used
for this work. Insertion of the foreign DNA into the beta-galactosidase coding sequence disables
the function of the enzyme so that colonies containing transformed DNA remain colorless
(white). Therefore, experimentalists are easily able to identify and conduct further studies on
transgenic bacterial clones, while ignoring those that do not contain recombinant DNA.