Molecular cloning is one of the basic techniques of genetic engineering. Molecular cloning is the method of isolating a single DNA fragment from a large mixture. Two cloning vectors, bacterial plasmids and phage (virus), have been most used. Each vector is grown on a bacterial cell and can accommodate foreign DNA without interfering with its replication ability.
If it is possible to clone an entire organism, it is also possible to clone a gene or a DNA fragment . This operation, is called molecular cloning.It is an important part of the activity conducted in research laboratories where molecular biology and genetics are practiced. It is based on a set of genetic engineering techniques that make it possible to isolate and reproduce genes.
Molecular Cloning And DNA
Antibiotic resistance genes of plasmids facilitate the isolation and identification of recombinant plasmids. Foreign DNA cleaved by the endonuclease Pst 1 when inserted into the single Pst 1 site of the plasmid’s ampicillin resistance gene acts as a mutation to the resistance gene. Since the plasmid also earned a functional tetracycline resistance gene, the plasmids can be recovered as clones by transfection into sensitive microbes grown on tetracycline-containing media. A single recombinant plasmid molecule gives rise to a microbial colony resistant to tetracycline but sensitive to ampicillin.
Thus, a restriction endonuclease DNA fragment is cloned. The method provides 1(T to 10 recombinant clones per ng of DNA. The recombinant DNA fragments are usually small (1 to 7,000 base pairs). Bacteriophage K provides a method for cloning larger DNA fragments (20,000 base pairs) at higher efficiency (10* to 10* recombinants . The middle portion (20,000 base pairs) of the 40,000 base pair bacteriophage is removed by restriction endonuclease and replaced with foreign DNA.
Recombinant bacteriophages are easily identified, since only those that are restored to approximately 40,000 base pair size can be packaged into viral particles, infect microbial cells, and form plaques (Fig. 34-2). Each plaque is the product of infection with a single molecule and thus contains a done of the foreign DNA fragment. The method permits molecular cloning of an individual’s genome in 10“ to 107 bacteriophage from digram quantities of DNA. More recently recombinantly designed cloning vectors that embody the advantage of plasmids and bacteriophage have been developed. These vectors, cosmids, clone 40,000 base pair sequences with high efficiency.
The human DNA used in molecular cloning is derived from either nuclear (genomic) or a DNA copy (cDNA) of messenger RNA synthesized in vitro. The strategy differs for isolation of recombinants of individual disease genes. Cloning of 3 hemoglobin, immunoglobulins, insulin, and growth hormone utilized cDNA cloning, since specialized cells were available where 10 to 90 per cent of the mRNA corresponded to the gene of Each of these methods permits isolation of a small fraction of the human genome (10* to 1(F) in a microbial vector. The recombination is easily amplified to milligram quantities of the gene fragment for detailed chemical study. The simplicity of the methods permits direct comparison of normal and individual patient’s disease genes.
Conclusion About Molecular cloning :
Molecular cloning involves isolating and purifying a gene or DNA fragment from a given organism. This gene or this purified DNA fragment is then introduced into another organism, most often unicellular (bacterium or yeast) or sometimes into a cell in culture from a multi-cellular organism. By dividing the bacterium, the yeast or the cell in culture will produce clones of itself which will each contain one or more copies of the gene or DNA fragment to be reproduced. This makes it possible to obtain a large number of copies of these DNA fragments of interest which one wishes to study.