Revision: Class 12 >> Biotechnology - Principles and Processes NEET (UG) Biotechnology - Principles and Processes

The technique of bringing about improvements in living organisms by genetic modifications and hybridization, for the welfare of human beings is known as ‘Biotechnology’.

The European Federation of Biotechnology (EFB) defined biotechnology as ‘the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.’

• Biotechnology, a term coined by Karl Ereky in 1919, is the use of biological systems and genetic modifications to develop products and services for human welfare.
• Traditional biotechnology relies on small-scale, natural processes like fermentation (e.g., producing curd and wine), whereas modern biotechnology operates on a large scale.
• Modern biotechnology is fundamentally driven by two core techniques: genetic engineering (the targeted alteration of DNA and RNA) and bioprocess engineering.
• The field experienced a major breakthrough with the development of recombinant DNA technology by Cohen and Boyer in 1973.
• By integrating disciplines such as molecular biology and biochemistry, biotechnology enables crucial applications in both medicine (antibiotics, vaccines, insulin) and agriculture (high-yield, disease-resistant crops).

• Two Core Techniques - Modern biotechnology is based on (i) Genetic Engineering and (ii) Chemical Engineering.
• Genetic Engineering - Deals with the alteration of DNA and RNA to achieve desired results in a directed, predetermined way using in vitro processes.
• Chemical Engineering - Maintains a sterile environment for manufacturing useful products like vaccines, antibodies, enzymes, vitamins, and therapeutics.
• What Genetic Engineering Involves - Repairing/replacing defective genes, synthesising new genes, transferring genes, combining genes from two organisms, and altering the genotype.
• Other Names for Genetic Engineering - Also called Recombinant DNA (rDNA) Technology or Gene Cloning, as it involves transferring a gene via a suitable vector to a new location or organism.

• The first recombinant DNA was created by Stanley Cohen and Herbert Boyer (1972) by linking the antibiotic resistance gene with a plasmid of Salmonella typhimurium.
• Restriction enzymes (molecular scissors) cut DNA at specific locations; ligase joins the cut ends to link the gene with the plasmid.
• Plasmids act as vectors to transfer the attached DNA into the host organism.
• Recombinant DNA transferred into E. coli replicated using the host's DNA polymerase — making multiple copies, called cloning.
• Steps in genetic modification - identify desirable genes → introduce into host → maintain and transfer DNA to progeny.

• Recombinant DNA technology manipulates and joins DNA from different sources using three main tools: enzymes, cloning vectors, and competent hosts.
• Restriction enzymes act as "molecular scissors" to cut DNA at specific sites, while DNA ligase acts as "glue" to seal the fragments together.
• Cloning vectors (such as plasmids or bacteriophages) serve as vehicles for carrying, inserting, and replicating foreign DNA within a host cell.
• Competent hosts are cells that have been specially treated (using techniques such as heat shock or gene guns) so they can successfully take up the foreign recombinant DNA.

• Restriction enzymes, often called "molecular scissors", are specialised endonucleases that cut DNA at specific internal positions to facilitate genetic engineering.
• Over 900 restriction enzymes, including the first discovered Hind II, have been successfully isolated from more than 230 strains of bacteria.
• These enzymes are named using a standard convention based on the genus, species, strain, and discovery order of the source bacterium, such as EcoRI.
• They function by inspecting DNA to find specific palindromic nucleotide sequences, which are base pair sequences that read the same on both strands in the same orientation.
• Type II restriction enzymes are the primary ones used in genetic manipulation because they reliably recognise and cut at very specific target sequences.
• Cutting the DNA slightly away from the centre of a palindrome creates "sticky ends", which are single-stranded overhangs that easily bond with complementary DNA using the enzyme DNA ligase.
• The resulting DNA fragments are separated by size using gel electrophoresis, where smaller negatively charged fragments move faster through an agarose matrix toward an anode.
• The separated DNA is visualised as bright orange bands under UV light after ethidium bromide staining and is carefully extracted from the gel through a process called elution.

• Vectors (like plasmids and bacteriophages) are DNA molecules used to carry and replicate foreign DNA inside a host cell.
• An ideal vector must have an origin of replication (ori), selectable markers, and specific cloning sites.
• pBR322 is a widely used standard bacterial plasmid vector containing these essential features.
• Recombinant DNA is identified using insertional inactivation (e.g., in blue-white selection, recombinant colonies appear white due to a disrupted gene).
• Higher organisms require specific vectors: Ti plasmids for plants and modified retroviruses for animals.

• DNA is hydrophilic, so it cannot enter cells easily; bacteria are made competent using Ca²⁺ ions.
• Cells are treated with cold (ice) and heat shock (42°C) to help the uptake of recombinant DNA.
• Transformation is the process of introducing recombinant DNA into bacterial cells.
• Microinjection → DNA is directly injected into the nucleus of animal cells.
• Biolistics (gene gun) and disarmed pathogens are used to transfer DNA into plant and host cells.

• Cells are first broken open using specific enzymes (such as lysozyme for bacteria) to successfully isolate the genetic material.
• The purified DNA is precisely cut at specific locations using restriction enzymes, which act as "molecular scissors" to extract the desired gene.
• The resulting DNA fragments are separated by size using gel electrophoresis, and the specific target sequence is extracted.
• The desired gene is then amplified into millions of copies using the Polymerase Chain Reaction (PCR) technique.
• The amplified gene is joined to a carrier vector using the enzyme DNA ligase to construct a new molecule called recombinant DNA.
• This recombinant DNA is introduced into a chemically treated, competent host cell (such as a bacterium) through a process known as transformation.
• For commercial use, these transformed host cells are cultured on a massive scale inside large, environmentally controlled vessels called bioreactors.
• The final therapeutic product undergoes downstream processing, which involves rigorous separation, purification, and quality testing before packaging.