CH-12
BIOTECHNOLOGY
Biotechnology
is defined as 'the development and utilization of biological forms, products or
processes for obtaining maximum benefits to man and other forms of life
• Biological agents means plants & animal cells, micro organisms, enzymes or their products .According to OECD (Organization for Economic Cooperation and Development, 1981)-'It is the application of scientific and engineering principles to the processing of materials by biological agents to provide goods and service to the human welfare'
.Biotechnology
occurs in two phases -
1.
Traditional (Old)Biotechnology
• It was based on fermentation technology using microorganisms as in the preparation of curd,ghee, soma, vinegar, yoghurt, cheese making, wine making etc.
• It
became an art of kitchen in Indian houses
2.Modern Biotechnology
• This
technique permits to change Or modify genetic material for getting new specific
products based on genetic engineering.
• It
includes rDNA technology, polymerase chain reaction (PCR), microarrays, cell
culture & fusion & Bioprocessing.
Principles and processes of Biotechnology
• Modern
biotechnology based on two core techniques genetic engineering and chemical engineering.
• Genetic engineering - It deals with alternation of genetic material (DNA or RNA).
• Chemical Engineering - It deals with maintaining sterile environment for manufacturing variety of useful products including vaccines, antibodies, enzymes, organic acids, vitamins, therapeutics etc.Genetic engineering is alternatively called recombinant DNA technology or gene cloning because -
1. Genetic
engineering involves manipulation of genes i. e. repairing of defective genes.
2. Replacing of
defective genes by healthy genes or normal genes.
3. Artificially
synthesizing of a totally new gene.
4. Transfer of genes
into a new location or into a new organism.
5. Introducing an all
together new genes.
•
Gene cloning -
In gene cloning, gene
of known function can be transferred from its normal location into a cell via a
suitable vector.
The transferred gene
is replicated normally and is handed over to the next progeny.
A. Tools and Technique for gene cloning/ rDNA technology :
The basic
requirements for this technique -
1) Different
instruments (devices) -
i. Macromolecules such as DNA, RNA & Proteins etc. are synthesized in the living cells which very in their molecular weight, solubility, presence of charges & absorbance of light.
ii. Several
techniques are used to isolate & characterize the macromolecules.
iii. The techniques are used on the basis of molecular weight, are gel permeation, osmotic pressure,ion exchange chromatography, spectroscopy, mass spectroscopy & eletrophoresis.
iv. Electrophoresis
is the separation of charged molecules, applying an electric field.
v. It is applied for
the separation ofof DNA, RNA & Proteins.
vi. DNA being
negatively changed, migrants to anode.
vii. Small fragments
of DNA molecule, move & separate fast.
viii. Use of Agarose
gel eletrophoresis, PAGE, SDA PAGE are the different methods of eletrophoresis.
• Polymerase
chain reaction (PCR) is another device used for gene cloning or gene
multiplication.
• It
is the the amplification of gene of interest through PCR.
• PCR generate a billion copies of the the desired segment of DNA or RNA, with high accuracy & specificity in few hours.
• The
device required for PCR is called thermal cyclar.
Requirements of PCR amplification
• DNA
containing the desired segment to be amplified.
• Several
molecules of four deoxyribonucleotide triphosphates (dNTPs)
• Excess
of two primer molecules
• Heat
stable DNA polymerase
• Mg++
ions.
Mechanism of PCR
• At
the start of PCR, the DNA segment, and excess of two primer molecules, four
deoxyribonucleosides triphosphates and thermostable DNA polymerase are mixed together in'eppendorf tube' and the following operations are performed sequentially.
•
Step I - Denaturation
The reaction mixture is heated to a temperature (90 -98°c) to seperate two strands of desired DNA is called Denaturation.
•
Step II - AnnealingAnnealing
The mixture is allowed to cool (40-60° c) that permits pairing of the primer to the complementary sequence in DNA is called annealing.
•
Step III - Extension
The temperature is raised to 70-75°c. TheTaq. polymerase adds dNTPs behind the primer on the SSDNA. It takes around two minutes duration.One cycle takes around 3-4 minutes.
• To begin second cycle, DNA is again
heated to convert double stranded DNA, into single strands.
• In an automatic thermal cycler, the
above three steps are automatically repeated 20-30 times.
• After it's sequencing, the amplified DNA
segment can be inserted into a cloning vector.
II) Biological tools
There are three types
of biological tools used for transformation with recombinant DNA.
a) Enzymes
Lysozymes, nucleases such as exonucleases, endonucleases, restriction endonucleases, DNA ligases,DNA polymerase, alkaline phosphates, reverse transcriptases etc.
• Nuclease : Enzymes that cut the phosphodiester bonds of polynucleotide chains are called nuclease.
• Nuclease
are of 2 types -
1. Exonuclease - Cut nucleotides from the
bottom ends of DNA strand.
2. Endonuclease - They make cuts at specific positions within the DNA. Bacteria nucleases that would recognize short nucleotide sequence with duplex DNA & cut.
i] Restriction endonuclease or Restriction enzymes
• The
phosphodiester back bone at highly specific sites on both strands of duplex is
cut by these enzymes called restriction enzymes.
• They
were given this name because they are used by bacteria to destroy various viral
DNAs that might enter the cell there-by restricting the growth potential growth
of the virus.
• Thus,
restriction enzymes serve as defence mechanism.
• The
restriction enzymes are thus the molecular scissors that are used to recognize
and cut DNA at specific sequences. The sites recognition sequences or
recognition sites.
• Different restriction enzymes found in different organisms recognize different nucleotide sequence & therefore cut DNA at different sites.
ii] Recognition sequences
• The sequence recognized by restriction enzymes are 4 to 8 nucleotides long and characterized by a particular type of internal symmetry.
• Consider
the particular sequence recognized by the enzyme ECORI.
3' ------ CTTAAG
------ 5'
5' ------ GAATTC ------
3'
When one reads the
sequence in opposite direction (3' to 5' or 5' or 3') it is identical/same.
• A
sequence with this type of symmetry is called Palindrome.
• When the enzyme EcoRI attacks this palindrome, it breaks each strand at the same site in the sequence, which is indicated by arrow between the A & G residues
3' ------- CTT AA G
------ 5'
5' ------- G AA TTC
------ 3'
Restriction enzyme either cut straight across the DNA in the region of palindrome to give blunt ends or cuts producing short, single stranded projection at each end of DNA of produce, cohesive or sticky ends or staggered ends.
B) Cloning Vectors - (vehicle DNA)
• Vectors
are DNA molecules that carry a foreign DNA segment and replicate inside the
host cell.
• Vectors may be plasmids, bacteriophages (M13, lambda virus) cosmid, phagemids, BAC (Bacterial Artificial Chromosome), YAC (yeast artificial chromosome), transposons, baculoviruses & mammalian artificial chromosome (MACs).
• Most
common used vectors are plasmid vectors - pBR 322, pUC, Ti plasmid
• A good vector should have ability of independent replication so that the vector replicate and large number of copies of DNA insert will be formed.
• A
vector should have marker genes for antibiotic resistance.
• They
must contain unique Cleavage site in one of the marker genes for restriction
enzyme.
• The plasmids obtained naturally do not posses all the characteristics. Hence they are constructed by inserting gene for antibiotic resistance. Ex. pBR 322, pBR 320, pACYC 177 – constructed plasmids.
• pBR 322 is mostly used in rDNA technology in plants..
i)
Plasmid
1. The plasmid used
in rDNA technology which replicate in E. Coli.
2. These plasmid is
used as vector in DNA cloning.
ii)
Plasmid vectors for plant
1. A plasmid vector
of new DNA is found in Agrobacterium tumefaciens.
2. Agrobacterium
found in soil & causes plant disease called crown gall (tumors).
3. A. tumefaciens
contains Ti plasmid (tumor-inducing) (Ti).
4. The Ti plasmid contains a transponson, called T DNA, which inserts copies of itself into chromosome of infected plant cells.
5. The transponson with new DNA, inserted into the host cells chromosome.
6. A plant cell containing this DNA, then grown in culture or induced to form a new, transgenic plant.
C)
Competent host (cloning organisms)
• Cloning
organisms is the bacteria like - Bacillus hemophilus, Helicobacter pyroli &
E. coli.
• Mostly
E. coli is used for transformation with recombinant DNA.
Methodology
for rDNA technology or Steps of gene cloning
a]
Isolation of DNA (gene) from the donor organisms
• The
desire gene has been obtained from donor organisms.
• The
cells of donor organisms are sheared with the blender & treated with
suitable detergent.
• Genetic
material from the donor is removed, isolated & purified by using several
techniques.
• Isolated
DNA can be spooled on to a glass rod.
• Isolated
purified DNA cut by restriction enzymes endonuclease.
• These enzymes cleave (cut) DNA at specific site called restriction sites & break DNA into fragments.
• Cleaved
DNA fragments have cohesive, sticky, staggered end or blunt ends.
• From cleaved DNA fragments, fragment containing desired gene is isolated and selected for cloning. This is now called foreign DNA or Passanger DNA.
• A
desired gene can be obtained directly from genomic library or cDNA library.
b] Insertion of desired foreign gene into a cloning vector (Vehicle DNA)
• The
foreign DNA is now inserted into vehicle DNA.
• The most commonly used cloning vectors are plasmid of bacteria (pBR 322) & bacteriophage (lambda phage & M13)
• Plasmids are isolated from bacterium using same restriction endonuclease (Plasmid i.e. vector DNA cleaved)
• By using DNA ligase, foreign DNA is inserted into vector DNA is called recombinant or chimeric DNA.
• Recombinant
DNA is transferred to bacterium is called transformation.
• The
transfer of rDNA into a bacterial cell is assisted by divalent Ca++.
• The
cloning organisms used in plant biotechnology are E. coli and Agrobacterium
tumifaciens.
• The
host/competent cell which has taken up rDNA is now called transformed cell.
• This is done by using techniques like - electroporation microinjection, lipofection, short gun,ultrasonification, biolistic method etc.
• But
in plant biotechnology the transformation is through Ti plasmids of A.
tumifaciens.
d]
Selection of the transformed host cell
• The transformation process generates a mixed population of transformed (recombinant) & nontransformed (non-recombinant) host cells.
• For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid vector is employed.
• For ex. pBR 322 plasmid vector contains different marker gene (Ampicillin restistant gene & Tetracycline resistant gene)
• When
pst1 RE is used, it knocks out Ampicillin resistant gene from plasmid, so that
the
recombinant cell
become sensitive to Ampicillin.
e]
Multiplication of transformed host cell
• Transformed host cells are separated by the screening process & introduced into fresh culture media.
• Host
cell devide & redivide with replication of rDNA.
f]
Expression of gene to obtain the desired product
• The
production of desired products like alcohol, enzymes & antibiotics.
• Finaly
desired products are separated, purified using suitable bioreactor.
