The techniques of using live organisms or enzymes from organisms to produce useful products and processes come under biotechnology.
The European Federation of Biotechnology (EFB) has given following definition of biotechnology:
The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services is called biotechnology.
Principles of Biotechnology
The two core techniques that enabled birth of modern biotechnology are:
- Genetic engineering: It involves the techniques to alter the chemistry of genetic material, techniques to introduce the altered genetic material into host organisms; in order to change the phenotype of the host organism.
- Maintenance of sterile (microbial contamination-free) ambience during the process so that only the desired microbe/eukaryotic cell is produced in large quantities.
Three basic steps in genetically modifying an organism are as follows:
- Identification of DNA with desirable genes;
- Introduction of the identified DNA into the host;
- Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.
Tools of Recombinant DNA Technology
Following are the key tools of recombinant DNA technology:
Restriction enzymes, polymerase enzymes, ligases, vectors, and the host organism
The enzymes which cut the DNA are called restriction enzymes. Hind II was the first restriction endonuclease to be discovered. Hind II always cuts DNA molecules at a particular point by recognizing a specific sequence of six base pairs. This specific base sequence is called the recognition sequence for Hind II. Today, more than 900 restriction enzymes are known. Different enzymes recognize different sequences.
Exonuclease: These enzymes remove nucleotides from the ends of the DNA.
Endonuclease: These enzymes make cuts at specific positions within the DNA.
Nomenclature of Restriction Enzymes: Each enzyme is named after the bacterium from which it was isolated. The naming system is based on bacterial genus, species and strain. Following table shows the name of EcoRI restriction enzyme and the implied naming system.
|Derivation of EcoRI name|
|I||First identified||Order of identification|
Palindromes: A group of letters that form the same word when read both forward and backward is called a palindrome. Restriction enzyme cut at the palindrome sequence in a DNA. Following is an example of palindrome sequence:
5' —— GAATTC —— 3'
3' —— CTTAAG —— 5'
Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands. This leaves single stranded portions at the ends. There are overhanging stretches called sticky ends on each strand. These are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.
When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of ‘sticky-ends’ and, these can be joined together (end-to-end) using DNA ligases.
Unless, the vector and the source DNA are cut with the same restriction enzyme, the recombinant vector molecule cannot be created.
Separation and isolation of DNA fragments: The cutting of DNA by restriction endonucleases results in the fragmentes of DNA. These fragments can be separated by a technique known as gel electrophoresis. Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix.
Agarose is the most commonly used matrix. It is a natural polymer which is extracted from sea weeds.
The DNA fragments separate according to their size through sieving effect provided by agarose gel. So, smaller fragments move farther than longer fragments.
The separated DNA fragments are stained with ethidium bromide. After that, exposure to UV radiation makes them visible. The DNA fragments appear as bright orange bands in this case.
The separated bands of DNA are cut out from agarose gel and extracted; constructing recombinant DNA by joining them with cloning vectors.
A small piece of DNA; taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes; is called a cloning vector.
The following are the features that are required to facilitate cloning into a vector.
- Origin of replication (ori): The sequence from where replication starts in the DNA is called the Origin of Replication (ori). When a piece of DNA is linked to this sequence, it can be made to replicate within the host cell. If you want to recover many copies of the target DNA, it should be cloned in a vector whose ‘ori’ supports high copy number.
- Selectable marker: These are the substances which help in identifying and eliminating non-transformants and selectively permitting the growth of the transformants. Generally, the genes which encode resistance to antibiotics (ampicillin, chloramphenicol, tetracycline, kanamycin, etc.) are considered useful selectable markers for E. coli. The normal E. coli cells do not carry resistance against any of these antibiotics.
- Cloning sites: All cloning vectors have recognition sites for the commonly used restriction enzymes. But to link the alien DNA, the vector needs to have very few, preferably single, recognition site. Ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.
Example: A foreign DNA can be ligated at the Bam H I site of tetracycline resistance gene in the vector pBR322. The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA but they can still be selected out from non-recombinant ones by plating the tranformants on ampicillin containing medium.
After that, the transformants (growing on ampicillin containing medium) are transferred on a medium which contains tetracycline.
The recombinant will grow in ampicillin containing medium but not in tetracycline containing medium.
But non-recombinants will grow in medium containing both the antibiotics. In this case, one antibiotic resistance gene helps in selecting the transformants, while other antibiotic resistance gene gets inactivated due to insertion of alien DNA. Thus, it helps in selection of recombinants.
Note: Selection of recombinants due to inactivation of antibiotics requires simultaneous plating on two plates having different antibiotics. Hence, it is a cumbersome process.
Alternate selectable markers have been developed which can differentiate recombinants from non-recombinants on the basis of their ability to produce color in the presence of chromogenic substrate.
In this case, a recombinant DNA is inserted within the coding sequence of an enzyme, α-galactosidase. This results into inactivation of the enzyme. (It is called insertional inactivation).
The presence of chromogenic substrate gives blue-colored colonies if plasmid in bacteria does not have an insert.
Presence of insert results into insertional inactivation of α-galactosidase, and no color is produced by the colonies. Such colonies are identified as recombinant colonies.
Vectors for cloning genes in plants and animals: Bacteria and viruses have been transferring their genes to transform eukaryotic cells; in order to force them to do what the bacteria or viruses want. Now, these tools of pathogens can be transformed into useful vectors for delivering beneficial genes to humans. For example; the tumor inducing (Ti) plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector which is no more pathogenic to the plants. This vector can now be utilized to transfer beneficial genes into many plants.
Competent Host (For Transformation with Recombinant DNA)
We know that DNA is a hydrophilic molecule. Hence, it cannot pass through cell membranes. In order to force bacteria to take up the plasmid, the bacterial cell needs to be ‘competent’’ to take up DNA. This is done by treating the bacterial cells with a specific concentration of a divalent cation, e.g. calcium. It increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
Recombinant DNA can then be forced into such cells in following steps:
- Cells along with recombinant DNA are incubated on ice.
- They are then briefly placed at 42°C (heat shock).
- Then they are put back on ice.
Micro-injection: In this method, recombinant DNA is directly injected into the nucleus of an animal cell.
Gene Gun: This method is applied for plant cells. In this method, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA.
Disarmed Pathogen: When the disarmed pathogen infects the cell, it transfers the recombinant DNA into host.