A major problem in biochemical research is obtaining sufficient quantities of
the substance of interest. These difficulties have been largely eliminated in
recent years through the development of molecular cloning techniques.
The clone is a collection of identical organisms that are all replicas of a
single ancestor.
Methods of creating clones of desired properties, usually called genetic
engineering and recombinant DNA technology, deserve much of the credit
for the dramatic rise of biotechnology since the mid-70'. The main idea of molecular
cloning is to insert a DNA segment of interest into an autonomously replicating
DNA molecule, called acloning vector, so that the DNA segment is replicated
with the vector. Such vectors could be, for instance, plasmids (circular DNAs
occurring in some bacteria). Reproduction of DNA segments in appropriate hosts,
results in the production of large amount of the inserted DNA segment.
A DNA to be cloned is usually a fragment of a genome of interest, obtained by
application of restriction enzymes. Most restriction enzymes cleave duplex DNA
at specific palindromic sites, and every two fragments have single strand
ends that are complimentary to each other (known as 'sticky ends'). Therefore,
a restriction fragment can be inserted into a cut made in a cloning vector by
the same restriction enzyme, because the segment ends stick (chemically bond)
to the loose ends of the vector. Such a recombinant DNA molecule is inserted
into a fast reproducing host cell, and is duplicated in the process of the host's
reproduction. The cells containing the recombinant DNA are then isolated from
non-infected cells using an antibiotic substance which the original vector is
resistant to. The cloning technique provides both high quantities of DNA fragments, as well
as a mean to preserve them for long periods of time (by keeping the host cells
alive).
