BIOTECHNOLOGY

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BIOTECHNOLOGY

• CHP.11 - Principles Process
• CLASS XII

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PRICIPLES OF BIOTECHNOLOGY

• (i) Genetic Engineering-to alter the chemistry of
  DNA RNA-introduced in host organism-change in
  phenotype of the host organism.
• (ii) Maintenance of sterile ambience.

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• Techniques of genetic engineering
• 1.Creation of recombinant DNA
• 2.Gene cloning
• 3.Gene transfer
• -alien piece of DNA attached with host chromosome
  at ORIGIN OF REPLICATION.
• -alien piece of DNA replicate and multiply itself
  in the host organism. CLONING)

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Construction of an artificial r-DNA molecule

• Stanley Cohen Herbert Boyer 1972-isolated
  antibiotic resistant gene and link it to PLASMID
  (self replicating circular extra-chromosomal DNA)
  of Salmonella typhimurium.
• Cutting of DNA at specific locations- restriction
  enzymes (molecular scissors).
• Cut DNA piece linked with plasmid DNA- these
  plasmid DNA act as VECTORS.
• Linking of antibiotic resistant gene with the
  plasmid vector-DNA Ligase enzyme.
• r-DNA transferred in E.coli a no. of copies
  formed by replication (CLONING)

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Three basic steps -

• 1.identification of DNA with desirable genes.
• 2.identified DNA introduced into host.
• 3.maintenence of introduced DNA in the host and
  transfer of the DNA to its progeny.

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TOOLS OF RECOMBINANT DNA TECHNOLOGY

• 1.Restriction Enzymes-
• restriction endonuclease- cut DNA at a particular
  point.
• First restriction endonuclease- Hind II.
• Exonucleases- remove nucleotides from the ends of
  DNA.
• Each restriction endonuclease recognises a
  specific palindromic nucleotide sequences in the
  DNA.
• Restriction enzymes cut the strand of DNA a
  little away from the centre of the palindromic
  site leaving single stranded streches called
  STICKY ENDS.
• Sticky ends are joined together by DNA ligase.

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Separation isolation of DNA fragments(Gel
Electrophoresis)

• DNA fragments are vely charged molecule.
• They are forced towards anode through Agarose
  matrix.
• Separated fragments are stained with Ethidium
  Bromide exposed to UV radiation.
• We can see bright Orange coloured bands of DNA.
• Separated bands of DNA are cut out from agarose
  gel extracted from gel piece.(ELUTION)
• Now DNA fragments are used in constructing r-DNA
  by joining them with cloning vectors..

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CLONING VECTORS

• (i)Origin of replication-(ori)-sequence from
  where replication starts,also responsible for
  controlling copy number of the linked DNA.
• (ii)Selectable marker-helps in identifying
  removing non transformants.
• (iii) Genes encoding resistance to antibiotics
  like Ampicillin,chloramphenicol,Tetracycline or
  Kanamycin are useful selectable markers for
  E.coli.

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Hind III
Cla I
EcoR I
Pvu I
tet R
BamH I
amp R
Pst I
pBR322
Sal I
Ori
rop
Pvu II
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(iv)Cloning Sites

• Ligation of alien DNA is carried out at a
  restriction site present in one of the two
  antibiotic resistance genes.
• EXAMPLE-A foreign DNA is ligated at the Bam H I
  site of tetracycline resistance gene in the
  vector pBR322.
• Recombinant plasmids lose tetracycline resistance
  can be selected from non-recombinants by
  plating the transformants on ampicillin
  containing medium then transferring on a medium
  containing tetracycline.
• Recombinants will grow in ampicillin containing
  medium but not on tetracycline medium.
• Non recombinants will grow on the medium
  containing both the antibiotics.

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• Presently alternative selectable markers are
  developed which differentiate recombinants from
  non recombinants on the basis of their ability to
  produce colour in the presence of a chromogenic
  substrate.
• A recombinant DNA is inserted within the coding
  sequence of an enzyme â-galactosidase. Enzyme
  gets inactivated. (INSERTIONAL INACTIVATION).
• Colonies do not produce any colour identified
  as recombinant colonies.

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(iv)Vectors for cloning genes in plants animals

• Agrobacterium tumifaciens a pathogen of dicot
  plants deliver T-DNA which transform normal plant
  cells into a tumor.
• The Tumor inducing plasmid (Ti-plasmid) of
  Agrobacterium is now modified into a CLONING
  VECTOR.
• RetroViruses in animals transform normal cells
  into Cancerous cells.Now they are disarmed used
  as CLONING VECTORS.

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Competent Host(for transformation with
Recombinant DNA)

• DNA is a hydrophillic molecule,it cannot pass
  through cell membranes.
• Bacterial cells are treated with divalent Cation
  (like Ca).
• Cells are incubated with r-DNA on ice, then
  placed briefly at 420C (Heat Shock) placed back
  on ice.
• MICROINJECTION-r-DNA is directly injected into
  the nucleus of an animal cell.
• BIOLISTICS (Gene Gun)-In plants cells are
  bombarded with high velocity micro-particles of
  Gold or Tungsten coated with DNA.

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Processes of Recombinant DNA Technology

• 1.Isolation of DNA- Cells are treated with
  enzymes such as LYSOZYME(Bacteria),
  CELLULASE(plant cells), CHITINASE (Fungi) to
  break the cell wall.
• RNA is removed by RIBONUCLEASE.
• Proteins are removed by PROTEASE.
• Purified DNA precipitates out after the addition
  of chilled ETHANOL.

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• 2.Cutting of DNA at specific locations
• By Agarose gel electrophoresis DNA undergoes
  restriction enzyme digestion.
• Cutting of Gene of interest addition of Ligase.
• Recombinant DNA is formed.

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3.Amplification of Gene of Interest using PCR

• PCR(Polymerase chain reaction)-
• Multiple copies of the gene of interest is
  synthesized in vitro using two sets of primers
  the enzyme DNA polymerase.
• Repeated amplification is achieved by using
  Taq-polymerase( which is isolated from a bacteria
  Thermus aquaticus) which remains active during
  the high temperature required for denaturation of
  double stranded DNA.

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• 4.Insertion of recombinant DNA into host
  Cell/organism.
• 5.Obtaining the foreign gene product-
• BIOREACTORS
• These are vessels in which raw materials are
  biologically converted to specific products,
  individual enzyme etc.
• It provides optimal conditions of growth
  (temperature, pH,substrate,salts,Vitamins,Oxygen
  ).

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DONSTREAM PROCESSING

• It includes separation purification of product.
• Product is formulated with suitable
  preservatives.
• Clinical trials of formulation.
• Strict quality control testing for each product.