RECOMBINANT DNA TECHNOLOGY
INTRODUCTION TO GENE CLONING AND GENETIC
Genetic recombination is a common phenomenon, usually observed in all
living organisms. Genetic recombination has been observed to occur during
normal sexual reproduction, which mainly consists of the breakage and
rejoining of deoxyribose nucleic acid (DNA) fragments of the chromosome.
This phenomenon is essential and useful in reassortment of genetic material.
Recombinant DNA techniques comprising of all the cloning steps is another
way of mimicking natural genetic recombination. Hence, the genetic
engineering and gene cloning offer potentially unlimited opportunities for
creating unique combinations of genes within a very short period of time, which
do not exist presently in the natural conditions.
Gene technology can be defined as a modification of the genetic properties of
an organism by the use of recombinant DNA technology.
In genetic engineering, the basic requirement is considered to be DNA. DNA
can be isolated from any cell such as plants, animal cells, bacteria, yeasts, etc.
They can be fragmented with the restriction endonucleases as we desire. Such
fragments can then be ligated to another piece of DNA plasmid vector.
Subsequently, they are further introduced by transformation into bacteria. The
host cell can be propagated in mass to characterise the genetic element such as
plasmid containing the gene of interest. After the plasmid extraction from the
recombinant bacteria, the plasmid DNA is further screened by PCR
(Polymerase Chain Reaction) by amplifying gene of interest and also by
restriction enzyme digestion in order to observe the release of the fragment
which was earlier ligated and transformed. They can also be further
characterised by southern blotting, by gene expression, SDS-polyacrylamide gel
electrophoresis, etc. The present text book deals with all the basic and essential
elements of cloning and gene expression (in prokaryotes and eukaryotes).
WHAT IS GENE CLONING?
Gene Cloning can be defined as construction of new recombinant plasmid
DNA molecule by following sequential steps such as restriction enzyme
digestion, ligation and transformation.
In a stepwise manner, it can be described as cutting the target gene, ligation of
target gene with a vector plasmid, transformation of the ligated product into E.
coli, followed by selection of right clones and finally amplification or
propagation of right clones.
Gene cloning is the process containing various number of steps as illustrated
in the Figure below:
The basic steps involved in gene cloning are as follows:
1. A DNA fragment coding for the gene of interest is cut and inserted into
the vector known as plasmid DNA. The resultant product is known as
recombinant DNA molecule.
2. Plasmid is known as vector, as it acts as a vehicle for the transport of the
gene into the host cell. The host cell can be bacterium, yeasts or animal cells.
3. After introduction of recombinant plasmid in the host cell, the carrier is
known as recombinant host. The plasmid through the process of replication
gets amplified to make large number of copies.
4. The recombinant plasmid is passed onto its progeny or F1 generation by
multiplication of the bacterium; the recombinant bacterium gets multiplied
5. After multiple divisions of bacterium, a clone of identical colonies is
produced. Each cell in the colony contains a single clone to multiple copies
of the recombinant plasmid.
RECOMBINANT DNA TECHNOLOGY :
Recombinant DNA technology, which is also called gene cloning or
molecular cloning, is a general term that encompasses a number of
experimental protocols leading to the transfer of genetic information
(DNA) from one organism to another.
There is no single set of methods that can be used to meet this objective;
however, a recombinant DNA experiment often has the following format.
• The DNA (cloned DNA, insert DNA, target DNA, or foreign DNA)
from a donor organism is extracted, enzymatically cleaved (cut, or
digested), and joined (ligated) to another DNA entity (a cloning vector) to
form a new, recombined DNA molecule (cloning vector– insert DNA
construct, or DNA construct).
• This cloning vector–insert DNA construct is transferred into and
maintained within a host cell. The introduction of DNA into a bacterial
host cell is called transformation.
• Those host cells that take up the DNA construct (transformed cells) are
identified and selected (separated, or isolated) from those that do not.
• If required, a DNA construct can be created so that the protein product
encoded by the cloned DNA sequence is produced in the host cell.
Various steps of Recombinant DNA Technology:
1. Isolation of gene
2. Preparation of target DNA
3. Insertion of DNA into plasmid
4. Insertion of plasmid back into cell
5. Plasmid multiplication
6. Target cells reproduction
7. Cells produce proteins.