NCERT Based KT’s PowerNotes NEET 2022

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Biotechnology Principle and Processes
What is Biotechnology? Action of Restriction nuclease
• Biotechnology deals with techniques of using live organisms or • Types
enzymes from organisms to produce products and processes  Exonucleases remove nucleotides from the ends of the DNA
useful to humans  Endonucleases make cuts at specific positions within the DNA
• Biotechnology involve genetically modified organism • Restriction endonuclease
• Example of Biotechnology processes : Test tube baby,  Restriction endonuclease recognises a specific palindromic
developing vaccine, correcting defective gene nucleotide sequence in the DNA → sticky ends are formed that
• Making curd, bread or wine, though microbial product but these can be joined by DNA ligase
are not part of biotechnology process  EcoRl recognition sequence is 5' GAATTC 3'
 When cut by the same restriction enzyme, the resultant DNA
Principles of biotechnology fragments have the same kind of ‘sticky-ends’ and, these can
Techniques of biotechnology: be joined together (end-to-end) using DNA ligases
• Genetic engineering Separation and isolation of DNA fragments
 Alter the Genetic material (DNA and RNA) → microbe is Agarose Gel electrophoresis
modified → changes the phenotype of organism
Motive Fragments cut by restriction endonuclease are separated by Gel
• Bioprocess engineering
electrophoresis
 Growing modified microbes in ambience → vaccine, enzyme Charge DNA fragments are negatively charge, move towards anode
etc is produced Separation Fragments are separated based on size, smaller moves farther
Traditional hybridisation procedures used in plant and animal Gel Gel is obtained from sea weed (red algae)
breeding, very often lead to inclusion of undesirable genes this Elution The separated bands of DNA are cut out from the agarose gel and
limitation is overcome by biotechnology which to allows only set extracted from the gel piece
of desirable genes into the target organism Visualize DNA Use ethidium bromide, followed by UV exposure --- orange colour
Sieving effect Provided by gel
Construction of First recombinant DNA Cloning vector
Cloning vectors produces multiple copies of desired gene
Stanley Cohen and Herbert Boyer isolated antibiotic resistance Vectors: Plasmid or bacteriophage (have high copy number)
gene from Salmonella Typhi by molecular scissors’– restriction
enzymes → The cut piece of DNA was then linked with the Features of cloning vector
plasmid (Vector) DNA by DNA Ligase → recombinant DNA is
introduced into E coli which multiplied using DNA polymerase 1) Origin of replication (ori)
enzyme → multiply copies of antibiotic resistance gene formed in • This is a sequence from where replication starts
E Coli (cloning) • ori responsible for controlling the copy number of DNA
• Foreign DNA linked to ori also replicates when vector DNA
Isolation of the Genetic Material (DNA) replicates
Component Digestive enzyme
2) Cloning sites
Bacteria Lysozyme
Plant cells Cellulase
• Vector preferably should have single recognition site for the
Fungus Chitinase
commonly used restriction enzymes
RNA Ribonuclease • Presence of more than one recognition sites within the vector
Proteins Protease will generate several fragments

3) pBR 322
• DNA is precipitate with chilled ethanol • Bollivar and Rodrigues synthesized pBR 322
• DNA that separates out can be removed by spooling • pBR322 is the plasmid and most used cloning vector of E. coli.
• Ampicillin (ampR) & tetracycline (tetR) are selectable markers.
Cutting of DNA at Specific Locations • It has ori and different cloning like BamHl in tetR ,
DNA is cut at specific location by restriction endonuclease Pvul and Pstl in ampR
Discovery of restriction endonuclease • rop codes for the proteins involved in the replication of plasmid.
• In 1963, Two enzymes were discovered from E Coli which
protect E Coli from bacterial infection (bacteriophage)
 Methylase : It adds methyl group to palindrome of E Coli
 Restriction endonuclease : Cut palindrome of virus
• First restriction endonuclease that was characterized – HIND II

Convention for naming restriction enzyme

Biology BOMB
 NCERT Based KT’s PowerNotes NEET 2022
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• Retrovirus is disarmed and then used to deliver desirable genes
4) Selectable marker into animal cells.
• Selectable marker, helps in identifying transformants and non Competent Host
transformants • Host cell must be made competent to take up DNA
• Antibiotic resistance genes are considered useful selectable • DNA is a hydrophilic molecule, it cannot pass through cell
markers for E. coli membranes host (bacterial cell)
• Antibiotic resistance gene allow the growth of only • Transformation is a procedure through which a piece of DNA is
transformants in the media introduced in a host bacterium
a) Antibiotic resistance gene 1) To Pass DNA into host cell
 Treat bacterial cell with divalent cation (calcium) → this
creates pores in cell wall→ recombinant DNA enters the cell
 Animal cell → micro-injection technique : recombinant DNA
is directly injected into the nucleus of an animal cell
 Plant cell → biolistic or gene gun (micro-particles of gold or
tungsten is used) : plants, cells are bombarded with high
velocity micro-particles of gold or tungsten coated with DNA
 Disarmed pathogen vectors like retrovirus having recombinant
DNA are used to infect the cell
2) To enable bacterial cell take up DNA
 Heat shock treatment enables bacteria to take up DNA
Antibiotic resistance gene are called as selection marker as it helps Obtaining the Foreign Gene Product
in selection of recombinants.
Heterologous expression refers to the expression of a gene in a
b) Beta galactosidase enzyme host organism which does not naturally have this gene
Beta galactosidase help differentiate recombinants from non- eg Insulin is not produce naturally by E Coli but if insulin gene is
recombinants on the basis of their ability to produce colour introduced into then E Coli starts producing Insulin so here E Coli
is called Heterologous host which produced recombinant protein
Bioreactor
Bioreactor is a vessel where large volumes (100-1000 litres) of
culture can be processed.
• A bioreactor provides the optimal conditions for desired product
• The most commonly used bioreactors are of stirring type
• Stirring type bioreactors are of two kinds:
Simple stirred tank bioreactor, Sparged stirred-tank bioreactor
Simple stirred tank bioreactor Sparged stirred-tank bioreacto
Has stirrer which helps in uniform mixing This has increased surface area for
c) Ampicillin resistance gene and availability of oxygen throughout the oxygen transfer than simple stirred-
if a recombinant DNA bearing gene for resistance to an antibiotic bioreactor. tank
(e.g., ampicillin) is transferred into E. coli cells, the host cells
Downstream Processing
become transformed into ampicillin-resistant cells. When
• Downstream processing includes Separation and purification
transformed cells are spread on agar plates containing ampicillin,
• Add preservative, do clinical trial, do quality control testing
only transformants will grow, untransformed recipient cells will
PCR
die. The ampicillin resistance gene in this case is called
• PCR stands for Polymerase Chain Reaction
a selectable marker.
• Multiple copies of the gene (or DNA) of interest is synthesized
5) Vectors for cloning genes in plants and animals • in vitro procedure
• Steps of PCR :
Vector Host cell (to which vector delivers a gene)
pbr 322 Bacterium Denaturation Separation of DNA strands : 95 0C
Agrobacterium Plant cell Annealing Joining of DNA primer : 55 0C
Retrovirus animal cell Primer is small chemically synthesised oligonucleotides
that are complementary to the regions of DNA
Also note : Human genome project use : BAC (bacterial Extension / Occurs at : 72 0C
artificial chromosomes)YAC (yeast artificial chromosomes). elongation Taq polymerase isolated from Thermus aquaticus is
Agrobacterium tumefaciens /polymerization used in this process
• It is a pathogen of Dicot plants
• It transfers its T DNA into plant cell and transform normal plant • Amplification : above steps of PCR can be repeated to make
cells into a tumorous cells billion copies of DNA, such repeated amplification is achieved
• Modified Ti plasmid (tumor inducing plasmid) is used as vector by the use of a thermostable DNA polymerase which remain
to deliver gene of interest in plant cell active during the high temperature induced denaturation of
double stranded DNA
Retrovirus • After 30 cycles, what began as a single molecule of DNA has
• Retroviruses have the ability to transform normal animal cells been amplified into more than a billion copies
into cancerous cells • Uses of PCR : Forensic study (DNA fingerprinting) ,
Biotechnology, HIV testing, Gene Therapy

Biology BOMB