The cornerstone of most molecular biology technologies is the gene. Basic to all biotechnology research is the ability to manipulate DNA. To facilitate the study of genes, they can be isolated and amplified.
One method of isolation and amplification of a gene of interest is to clone the gene by inserting it into another DNA molecule that serves as a vehicle or vector that can be replicated in living cells. When these two DNAs of different origins are combined, the result is a recombinant DNA molecule.
Although genetic processes such as crossing-over technically produce recombinant DNA, the term is generally reserved for DNA molecules produced by joining segments derived from different biological sources. The recombinant DNA molecule is placed in a host cell, either prokaryotic or eukaryotic. The host cell then replicates (producing a clone), and the vector with its foreign piece of DNA also replicates. The foreign DNA thus becomes amplified in number.
Historical Perspective of Recombinant DNA Technology
In the early 1960s, before the advent of gene cloning, studies of genes often relied on indirect or fortuitous discoveries, such as the ability of bacteriophages to incorporate bacterial genes into their genomes.
The synthesis of many disparate experimental observations into recombinant DNA technology occurred between 1972and 1975, through the efforts of several research groups working primarily on bacteriophage lambda (λ).
One of the first recombinant DNA molecules to be engineered as a hybrid of phage λ and the SV40 mammalian DNA virus genome. In 1974 the first eukaryotic gene was cloned. Amplified ribosomal RNA (rRNA) genes or “ribosomal DNA” (rDNA) from the South African clawed frog Xenopus laevis were digested with a restriction endonuclease and linked to a bacterial plasmid. Amplified rDNA was used as the source of eukaryotic DNA since it was well characterized at the time and could be isolated in quantity by CsCl-gradient centrifugation.
Within oocytes of the frog, rDNA is selectively amplified by a rolling circle mechanism from an extrachromosomal nucleolar circle. The number of rRNA genes in the oocyte is about 100- to 1000-fold greater than within somatic cells of the same organism. To the great excitement of the scientific community, the cloned frog genes were actively transcribed into rRNA in E. coli. This showed that recombinant plasmids containing both eukaryotic and prokaryotic DNA replicate stably in E. coli. Thus, genetic engineering could produce new combinations of genes that had never appeared in the natural environment, a feat that led to widespread concern about the safety of recombinant DNA work.
Making a Recombinant DNA
How does recombinant DNA technology work? The organism under study, which will be used to donate DNA for the analysis, is called the donor organism. The basic procedure is to extract and cut up DNA from a donor genome into fragments containing from one to several genes and allow these fragments to insert themselves individually into opened-up small autonomously replicating DNA molecules such as bacterial plasmids. These small circular molecules act as carriers, or vectors, for the DNA fragments. The vector molecules with their inserts are called recombinant DNA because they consist of novel combinations of DNA from the donor genome (which can be from any organism) with vector DNA from a completely different source (generally a bacterial plasmid or a virus).
The recombinant DNA mixture is then used to transform bacterial cells, and it is common for single recombinant vector molecules to find their way into individual bacterial cells. Bacterial cells are plated and allowed to grow into colonies. An individual transformed cell with a single recombinant vector will divide into a colony with millions of cells, all carrying the same recombinant vector. Therefore an individual colony contains a very large population of identical DNA inserts, and this population is called a DNA clone.
A great deal of the analysis of the cloned DNA fragment can be performed at the stage when it is in the bacterial host. Later, however, it is often desirable to reintroduce the cloned DNA back into cells of the original donor organism to carry out specific manipulations of genome structure and function.
Application of Recombinant DNA Technology
The techniques for gene manipulation, cloning, and expression were first developed in bacteria but are now applied routinely in a variety of model eukaryotes. The genomes of eukaryotes are larger and more complex than those of bacteria, so modifications of the techniques are needed to handle the larger amounts of DNA and the array of different cells and life cycles of eukaryotes.
The possibility of transgenic modification of eukaryotes such as plants and animals (including humans) open up many new approaches to research because genotypes can be genetically engineered to make them suitable for some specific experiment. A particularly exciting application of transgenesis is in human gene therapy the introduction of a normally functional transgene that can replace or compensate for a resident malfunctioning allele. Because of their economic significance, plants have long been the subject of genetic analysis aimed at developing improved varieties.
Recombinant DNA technology has introduced a new dimension to this effort because the genome modifications made possible by this technology are almost limitless. No longer is breeding confined to selecting variants within a given species. DNA can now be introduced from other species of plants, animals, or even bacteria.
In Plants
Another area of great promise is “molecular farming,” making transgenic plants that contain health-care products such as pharmaceuticals and vaccines. Plants containing vaccines are particularly important for developing countries because the vaccines are inexpensive, orally administered without specialized techniques (possibly eating one seed is all that is necessary), and easy to deliver to remote areas (vaccines currently require refrigeration and often decay en-route)
In Animals
There are several ways of producing transgenic animals. Mice are the most important models for mammals generally. Furthermore, much of the general technology developed in mice can be applied to humans. Two key techniques are the ability to disrupt a gene (perhaps for reverse genetics study) and to replace one allele with another. Gene disruptions are sometimes called knockouts. An organism carrying the gene knockout can then be examined for altered phenotypes. Knockout mice are invaluable models for the study of mutants similar to those found in humans.
In Gene Therapy
The general approach of gene therapy is nothing more than an extension of the technique for clone selection by functional complementation. The functions absent in the recipient as a result of a defective gene are introduced on a vector that inserts into one of the recipient’s chromosomes and thereby generates a transgenic animal that has been genetically “cured.” The technique is of great potential in humans because it offers the hope of correcting hereditary diseases. However, gene therapy is also being applied to mammals other than humans.
Human Gene Therapy
Perhaps the most exciting and controversial application of transgenic technology is in human gene therapy, the treatment and alleviation of human genetic disease by adding exogenous wild-type genes to correct the defective function of mutations.
Two basic types of gene therapy can be applied to humans, Germline and Somatic.
The goal of germ-line gene therapy is more ambitious i.e., to introduce transgenic cells into the germline as well as into the somatic cell population. Somatic gene therapy focuses only on the body (soma). The approach is to attempt to correct a disease phenotype by treating some somatic cells in the affected person.
Usage of Recombinant DNA Technology in Commercial and Therapeutic Applications
- Therapeutic Cloning
Therapeutic Cloning is performed to harvest embryonic stem cells. These cells are found inside the developing embryo and they can be used to develop tissues & organs. A cell is removed from the patient’s body. Its nucleus is taken out and inserted into an enucleated egg cell. Cell division is triggered either chemically or through electric shock. The resulting embryonic stem cells are then removed and used to treat the patient. Therapeutic Cloning can help in tissue or organ transplantation.
- Reproductive Cloning
In reproductive cloning, the ovum of the animal to be cloned is enucleated. Then a cell is taken from the same organism and its nucleus is removed. This nucleus is then transferred into the enucleated egg cell.
- Human cloning
Even after the revelation of the entire human genome, our understanding of human genome is quite trivial. Only 1.5% of the entire genome contains genes. The rest, overlooked so far as ‘junk DNA’, may have also a bearing on the expression of genes.
