Cloning

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 occuring 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, generally two fragments that have single strand ends that are complimentary with 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 .