Introduction
Q. What is Biotechnology?
Ans: Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans. Making curd, bread or wine, which are all microbe-mediated processes, could also be thought as a form of biotechnology. However, it is used in a restricted sense today, to refer to such of those processes which use genetically modified organisms to achieve the same on a larger scale.
Modern biotechnology using genetically modified organisms was made possible only when man learned to alter the chemistry of DNA and construct recombinant DNA. This key process is called recombinant DNA technology or genetic engineering. This process involves the use of restriction endonucleases, DNA ligase, appropriate plasmid or viral vectors to isolate and ferry the foreign DNA into host organisms, expression of the foreign gene, purification of the gene product, i.e., the functional protein and finally making a suitable formulation for marketing. Large-scale production involves use of bioreactors.
Biotechnology
- Genetic engineering involves the techniques to alter the chemistry of genetic material (DNA and RNA) and thus change the phenotype of the host organism.
- Asexual reproduction preserves the genetic information, while sexual reproduction permits variation.
- Traditional hybridization procedures used in plant and animal breeding, very often lead to inclusion and multiplication of undesirable genes along with the desired genes.
- The techniques of genetic engineering which include the creation of recombinant DNA, use of gene cloning and gene transfer, overcome this limitation and allows us to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into the target organism.
- There are three basic steps in genetically modifying an organism — 1. identification of DNA with desirable genes 2. introduction of the identified DNA into the host 3. maintenance of introduced DNA in the host and transfer of the DNA to its progeny.
- DNA which is somehow transferred into an alien organism would not be able to multiply itself in the progeny cells of the organism.
- But, when it gets integrated into the genome of the recipient, it may multiply and be inherited along with the host DNA. This is because the alien piece of DNA has become part of a chromosome, which has the ability to replicate.
- In a chromosome there is a specific DNA sequence called the origin of replication, which is responsible for initiating replication.
- Therefore, for the multiplication of any alien piece of DNA in an organism it needs to be a part of a chromosome(s) which has a specific sequence known as ‘origin of replication’.
- Thus, an alien DNA is linked with the origin of replication, so that, this alien piece of DNA can replicate and multiply itself in the host organism. This can also be called as cloning or making multiple identical copies of any template DNA.
- Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.
- Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure. They differ only in the nucleotide sequence within that identical overall structure.
- In most cases, organisms containing recombinant DNA have apparently normal phenotypes. That is, their appearance, behavior, and metabolism are usually unchanged.
- The cutting of DNA at specific locations became possible with the discovery of the so-called ‘molecular scissors’- restriction enzymes.
- Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two kinds exonucleases and endonucleases.
- Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make cuts at specific positions within the DNA.
- The cut piece of DNA was then linked with the plasmid DNA. These plasmid DNA act as vectors to transfer the piece of DNA attached to it.
- You probably know that mosquito acts as an insect vector to transfer the malarial parasite Into human body.
- In the same way, a plasmid can be used as vector to deliver an alien piece of DNA into the host organism.
- The linking of antibiotic resistance gene with the plasmid vector became possible with the enzyme DNA ligase, which acts on cut DNA molecules and joins their ends. This makes a new combination of circular autonomously replicating DNA created in vitro and is known as recombinant DNA.
- When this DNA is transferred into Escherichia coli, a bacterium closely related to Salmonella, it could replicate using the new host’s DNA polymerase enzyme and make multiple copies. The ability to multiply copies of antibiotic resistance gene in E. coli was called cloning of antibiotic resistance gene in E. coli.
Image: Biotechnology
Source: NCERT Text Books
- Recombinant human insulin,
- Recombinant human growth hormone,
- Recombinant blood clotting factor VIII,
- Recombinant hepatitis B vaccine,
- Insect-resistant crops etc.
- You may be surprised to know that we have learned the lesson of transferring genes into plants and animals from bacteria and viruses which have known this for ages - how to deliver genes to transform eukaryotic cells and force them to do what the bacteria or viruses want.
- For example, Agrobacterium tumefacient, a pathogen of several dicot plants is able to deliver a piece of DNA known as ‘T-DNA’ to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen.
- Similarly, retroviruses in animals have the ability to transform normal cells into cancerous cells.
- A better understanding of the art of delivering genes by pathogens in their eukaryotic hosts has generated knowledge to transform these tools of pathogens into useful vectors for delivering genes of interest to humans. •
- The tumor-inducing (Ti) plasmid of AgrobcLCterium tumefacient has now been modified into a cloning vector which is no more pathogenic to the plants but is still able to use the mechanisms to deliver genes of our interest into a variety of plants.
- Similarly, retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells.
- So, once a gene or a DNA fragment has been ligated into a suitable vector it is transferred into a bacterial, plant or animal host (where it multiplies).
- Plasmids and bacteriophages [vectors] have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.
- Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. In order to force bacteria to take up the plasmid, the bacterial cells must first be made ‘competent’ to take up DNA.
- Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 420C (heat shock) and then putting them back on ice. This enables the bacteria to take up the recombinant DNA. This is not the only way to introduce alien DNA into host cells.
- In a method known as micro-injection, recombinant DNA is directly injected into the nucleus of an animal cell.
- In another method, suitable for plants, cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA in a method known as biolistics or gene gun.
- And the last method uses ‘disarmed pathogen’ vectors, which when allowed to infect the cell, transfer the recombinant DNA into the host.