NEET Biology Notes Biotechnology Principles and its Applications Biotechnology
Biotechnology is the use of organisms, their parts or processes for the manufacture or production of useful or commercial substances and for the provision of services such as waste treatment.The definition of biotechnology given by European Federation of Biotechnology (EFB) is that the integration of natural science and organisms, cells, parts there of and molecular analogues for products and services. Microorganisms are particularly suitable for industrial processes due to their easy and fast growth and higher yield and more specific products.
Principles of Biotechnology
The two core techniques that enabled the birth of modern biotechnology are
- Genetic engineering is the technique of altering the nature of genetic material and/or introduction of it into the host organisms to change its phenotype.
- Techniques to facilitate the growth and multiplication of only the desired microbes or cells in large number under sterile conditions for the manufacture of biotechnological products.
The techniques of genetic engineering include:
- Creation of recombinant DNA (rDNA)
- Use of gene cloning
- Gene transfer
Recombinant DNA Technology
Recombinant DNA technology, commonly known as genetic engineering is the most useful technique for creating deliberate modification of an organisms genome or particular gene. This technology is the foundation of biotechnological revolution. Through this technology, it has become possible to transfer genes between distantly related organisms such as humans and bacteria or plants and bacteria. If these genes integrate permanently. into the desired organism, they can be transferred to offspring and the resulting organism is said to be transgenic or recombinant organism.
Tools of Recombinant DNA Technology
The main tools of recombinant technology are
- Vector (s)
- Host organism/cell.
Restriction endonucleases, polymerases and ligases are the main enzymes which are used in genetic engineering.
Restriction endonucleases Restriction endonucleases cut a DNA molecule within certain specific sites that have specific base sequence.
Therefore, these are also known as molecular scissors Restriction enzyme Hae III cuts DNA, where ever it recognizes the sequence cut is made
between adjacent G and C. The restriction enzymes Eco RI, Bam II and Hind III are used in recombinant DNA technology to produce cuts at specific site of desired DNÀ.
Some important facts regarding restriction enzymes are as follows :
- The first restriction endonuclease isolated was Hind III.
- About 900 restriction enzymes are known that have been isolated from 230 strains of bacteria.
- Restriction enzymes cut DNA into pieces.
- Typical restriction site is 4-6 nucleotide base pairs (bp) long.
- Restriction enzymes are palindromes, i.e. they read the same forwards as well as backwards (e.g. bob, racecar)
Ligases help in sealing gaps in DNA fragments by forming phosphodiester bonds. These are also known as molecular glue or binder, e.g.T4DNA ligase.
Polymerases are used in synthesising copy of DNA on complementary DNA, e.g. DNA polymerase, reverse transcriptase.
Alkaline phosphatases cut-off phosphate group from end of linearised circular DNA to check its recircularisation.
Cloning Vectors : Vehicles for Cloning
The vectors are DNA molecules that can carry a foreign DNA segment and replicate inside the host cell. Vectors may be plasmids, bacteriophage, cosmids, phagemids, Yeast Artificial Chromosomes (YAC), Bacterial Artificial Chromosomes (BAC) etc.
The following features are required to facilitate cloning in a vector:
- Origin of replication (Ori)
- Selectable marker
- Cloning of recognition site
- Small size of vector.
Plasmids are extrachromosomal, self-replicating, usually circular, double-stranded DNA molecules, found naturally in many bacteria and also in some yeast. The plasmid molecules may be present in 1 or 2 copies or in multiple copies (500-700) inside the host organism. These naturally occurring plasmids have been modified to serve as vectors in the laboratory. pBR 322 is an ideal plasmid sector and easily manipulated. It is the first artificial cloning vector constructed in 1977 by Boliver and Rodriguez.
- In the name P signifies plasmid, B and R are the two initials of the scientists who developed it.
- It contains origin of replication (ori) which allows production of multiple copies per cell.
- has the two selectable markers (antibiotic resistance genes) : tetracycline, tetR and ampicillin ampR
- also possesses unique recognition sites or cloning sites for 12 restriction enzymes (endonucleases). Two unique sites, Pst I and Pvu I are located withing the amp gene and Bam HI, Sal I with in tet gene, etc.
For preparing a genomic library of an eukaryote with a quite large DNA fragments or even whole genomes, the requirements of cloning are fulfilled by lambda phage derivatives used for transferring the genetic material from one bacterium to other. From these, cloning of 20-25 kb is possible.
These consist of linear double stranded DNA molecules, which have been engineered in the way that their lytic cycle is possible but lysogenic cycle is not possible. The lambda phage genome is of about 50 kbp circular DNA. It follows either a lytic path or a lysogenic path. Lytic path may be switched towards the lysogeny and vice-versa.
The lambda cloning vectors are of two types:
- The insertion vector, accepts inserts only 12 kbp long at a single multiple cloning site
- The replacement vector, which accepts inserts 9-23 kbp long with the involvement of replacement of a non-essential part (stuffer) of genome, e.g. EMBL3 and EMBL4 vectors.