Biotechnology is one of the most exciting emerging fields of applied biology that has remarkably impacted human life and expanded on its sophistication, scope, and applicability in recent years. The term biotechnology involves the use of biological processes, organisms, or systems to manufacture products intended to improve the quality of human life. It takes into account the various techniques used in the manipulation of DNA. This procedure for the several authors have attempted defining what biotechnology is, however one simple definition that conveys its meaning is that it is the commercialization of cell and molecular biology.
Biotechnology can generally be defined as "the use of living organisms, cells or cellular components for the production of compounds or precise genetic improvement of living things for the benefit of man". Classical biotechnology, though has been in practice for centuries now, the recent technological advances of the twentieth century, in the various branches of sciences such as physics, chemistry, as well as engineering, computer application, and information technology have revolutionized the development of life sciences, which ultimately resulted in the evolution of modern biotechnology. Modern biotechnology takes advantage of the basic biological sciences such as genetics, animal cell culture, histology, cell biology, microbiology, biochemistry and molecular biology along with other branches of science like chemical engineering, and information technology.
The twentieth century evolution of various arrays of biochemical, biophysical and molecular techniques has improved the quality of human life tremendously, leading to the development of new drugs, quickly and reliably diagnosis of diseases, production of new vaccines, development of food products, cosmetics, and industrially useful chemicals. In addition, genetically-altered crop plants, which can resist the stress of pests, diseases, and hash environmental conditions, have been successfully developed. New tools and techniques to extend the studies on genomics and proteomics, not only of man but other organisms have been developed. The recent involvement of information technology and internet in the field of biotechnology, particularly genomics and proteomics, has lead to the birth of the newly emerging field of bioinformatics and computational biology.
GENETIC ENGINEERING AND RECOMBINANT DNA TECHNOLOGY
Genetic engineering, otherwise known as recombinant DNA technology is the bedrock of the modern biotechnology. There are fundamental changes occurring in our society today due to rapid advances in this newly emerging field of science. Genetic engineering involves the technique of removing, modifying or adding genes to a DNA molecule in order to change the information it contains.
In general, all organisms are made up of the same type of genetic material (Adenine, Guanine, Thymine and Cytosine), biotechnologists now use enzymes to cut and remove DNA segments from one organism and then recombine it with DNA in another organism. This technology is called recombinant DNA technology. Recombinant DNA technology involves the creation of new combinations of DNA segments that are not found together in nature. This systematic isolation and manipulation of genes allows for more precise genetic analysis as well as practical applications in medicine, agriculture and industry.
RECOMBINANT DNA TECHNIQUES
The techniques of recombinant DNA encompass a number of methodologies or tools that enable us to construct new combinations of DNA(recombinant DNA or rDNA) in the laboratory for different purposes. The rDNA molecule thus constructed can be introduced into an appropriate host cell, where it can be multiplied and generate many copies. This forms the basic concept of the process known as gene cloning or DNA cloning. The goal of recombinant DNA is gene cloning to generate large amounts of pure DNA that can be manipulated and studied.
The following are the basic techniques of recombinant DNA technology involved in the process of creating recombinant DNA molecules (DNA cloning).
i. Methods of locating specific DNA sequence.
ii. Techniques for cutting DNA at precise location.
iii. Procedures at amplifying a particular DNA sequence billion of times, producing enough copies of a DNA sequence to carryout further manipulations.
iv. Methods for mutating and joining DNA fragments to produce desired sequence.
v. Procedures for transferring DNA sequences into recipient cells.
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STEPS IN RECOMBINANT DNA TECHNOLOGY
LOCATING AND ISOLATING THE SPECIFIC DNA SEQUENCE
The first and most formidable challenge is to locate the desired gene and separate it from the rest of the DNA. After locating the specific desired gene, then isolating the gene will enable the determination of its nucleotide sequence. From this information, the internal landmarks of the gene (such as intron number and position) can be determined.
A comparison of DNA sequences between genes also can lead to insights in gene evolution. Converting the DNA sequence of a gene into amino acid sequence by using the genetic code leads to comparisons with the protein products of known genes; and, from this knowledge, the function of the gene can often be interfered. Function can also be studied by direct modification of part of the gene's DNA sequence followed by the reintroduction of the gene into the genome. Furthermore, a gene can be moved from one organism to another. An organism containing a foreign gene is called transgenic. Transgenic organisms can either be used for basic research or for specialized commercial applications.
CUTTING AND JOINING DNA FRAGMENTS
Restriction enzymes are the draft horses of recombinant DNA technology and are used whenever DNA fragments must be cut or joined. The discovery of restriction enzymes in 1960s made the development of recombinant DNA technology a reality. Restriction enzymes otherwise known as restriction endonucleases recognize and make double-stranded cuts in the sugar-phosphate backbone of DNA molecules at specific nucleotide sequences. These enzymes are produced naturally by bacteria, where they are used in defense against viruses.
In bacteria, restriction enzymes recognize particular sequence in viral DNA and then cut it. A bacterium protects its own DNA from a restriction enzyme by modifying the recognition sequence, usually by adding methyl group to its DNA.
VIEWING DNA FRAGMENTS
Electrophoresis is a useful technique in separating the sliced DNA fragments after the completion of a restriction reaction. Electrophoresis is a standard biochemical technique for separating molecules on the basis of their size and electric charge.
Gel Electrophoresis: There are several types of electrophoresis, however to separate DNA molecules, gel electrophoresis is often used. Gel electrophoresis is widely used in recombinant DNA technology. It is often employed when there is a need to determine the number or size of DNA fragments or to isolate DNA fragments by size. A porous gel is often made from agarose (a polysaccharide isolated from seaweed), which is melted in buffer solution and poured into a plastic mold. As it cools, the agarose solidifies, making a gel that looks something like stiff gelatine.
Small indentions called wells are made at one end of the gel to hold solutions of DNA fragments, and electrical current is passed through the gel. Because the phosphate of each DNA nucleotide carries a negative charge, the DNA fragments migrate towards the positive end of the gel. In this migration, the gel acts as a sieve; as the DNA molecules migrate towards the positive pole, they move through the pores between the gel particles. Small DNA fragments migrate more rapidly than large ones and, with time, the fragments separate based on their size.
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