BIOTECHNOLOGY: PRINCIPLES AND PROCESSES
BIOTECHNOLOGY: PRINCIPLES AND PROCESSES
· Two core techniques that enabled birth of modern biotechnology:
o Genetic engineering: Techniques to alter the chemistry of genetic material (DNA and RNA) to introduce into host organisms and thus change the phenotype of the host organism.
o Maintenance of sterile (microbial contamination-free) ambient chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities.
Conceptual development of the principle of genetic engineering:
· Asexual reproduction preserves the genetic identity of species.
· Sexual reproduction creates variation and creates unique combinations of genetic makeup.
· Traditional hybridization procedures used in plant and animal breeding lead to inclusion of undesirable genes along with desired genes.
· The techniques of genetic engineering which includes 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 target organism
· Three basic steps in genetically modifying an organism –
o Identification of DNA with desirable gene
o Introduction of the identified DNA into the host.
o Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.
TOOLS OF RECOMBINANT DNA TECHNOLOGY:
· In the year 1963 two enzymes discovered from Escherichia coli which restrict the growth of bacteriophage in it.
o One of these added methyl groups to DNA.
o Other cut the phage DNA. (restriction endonuclease)
· The first restriction endonuclease discovered is Hind II.
· Hind II always cut DNA molecule at particular point by recognizing a specific sequence of six base pairs. This is called recognition sequence for Hind II.
· Till date around 900 restriction enzymes isolated from 200 strains of bacteria each of which recognize different recognition sequences.
· Restriction enzyme belongs to nucleases.
· There are two kind of nucleases:
· Exonuclease removes nucleotides from the free ends of the DNA.
· Endonucleases make cuts at specific positions within the DNA.
· Each restriction endonuclease recognizes a specific palindromic nucleotide sequences in the DNA.
· Palindromes are the group of letters that read same both forward and backward, e.g. “MALAYALAM”.
· The palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept same.
· The restriction enzyme cut the strand of DNA little away from the centre of the palindrome sites, but between the same two bases on the opposite strand. This leaves single stranded portions at the ends. There are overhanging stretches called sticky ends on each strand.
· This stickiness of the ends facilitates the action of the enzyme DNA ligases.
· The foreign DNA and the host DNA cut by the same restriction endonuclease, the resultant DNA fragments have the same kind of ‘sticky-ends’ and these can be joined together using DNA ligases.
Convention for naming restriction endonuclease:
· The first letter of the name comes from the genus.
· Second two letters come from the species of the prokaryotic cell from which the enzyme isolated
· The fourth letter is in capital form derived from the Strain of microbes.
· The Roman letter followed is the order of discovery
· Best example: EcoRI comes from Escherichia coli RY 13
Separation and isolation of DNA fragments:
· The cutting of DNA by restriction endonucleases results in the fragments of DNA.
· These fragments are separated by a technique called gel electrophoresis.
· Since the DNA fragments are negatively charged, they can be separated by forcing them to move towards anode under an electric field through a medium/matrix.
· Most commonly used matrix is agarose, a natural polymer extracted from sea weed.
· DNA fragments separate according to their size through sieving effect provided by the agarose gel. Hence the smaller the fragment size, farther it moves.
· The separated fragments are visualized by staining them with Ethidium bromide followed by exposure to UV radiation.
· The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is called elution.
· The plasmid and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.
· Alien DNA linked with the vector multiply its number equal to the copy number of the plasmid or bacteriophage.
Features of cloning vector:
Origin of replication:
· This is the sequence where the replication starts called ori gene.
· The alien DNA linked with vector also replicates.
· Controls the copy number of the linked DNA.
· It is required to identify recombinant from the non-recombinant.
· Helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants.
· Transformation is a procedure through which a piece of foreign DNA is introduced in a host bacterium.
· Normally, the gene coding resistance to antibiotics such as ampicilin. Tetracycline, chloramphenicol or kanamycins etc are considered as useful selectable markers for E.coli.
· Thr normal E.coli cells do not carry resistance against any of antibiotics.
Alternative selectable marker:
Vectors for cloning genes in plants and animals:
Competent Host (for transformation with recombinant DNA)
PROCESS OF RECOMBINANT TECHNOLOGY:
Isolation of the Genetic material (DNA):
Amplification of Gene of Interest using PCR:
Obtaining the Foreign Gene product or Recombinant product:
Biotechnology has wide applications in the field of medicine like genetically engineered insulin, gene therapy and molecular diagnosis. Transgenic animals are also the result of biotechnology. Bt cotton was also generated with the help of biotechnology.