Notes-Part-1-Class-12-Biology-Chapter-12-Biotechnology-Maharashtra Board

Biotechnology

Maharashtra Board-Class-12th-Biology-Chapter-12

Notes-Part-1

Topics to be Learn : Part-1

  • Biotechnology
  • Principles and Processes of Biotechnology
  • Methodology for r-DNA technology

Topics to be Learn : Part-2

  • Applications of Biotechnology
  • Bioethics
  • Effects of Biotechnology on the Environment
  • Effects of Biotechnology on Human Health
  • Biopatent and Biopiracy

Biotechnology :

  • The term biotechnology was first used by Karl Ereky in 1919 to describe a process for large scale production of pigs.
  • 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’
  • 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’.
  • Scientific and engineering principles : Scientific principles of microbiology, genetics biochemistry, chemical engineering, mathematics, statistics, computers, industrial processes, etc.
  • Biological agent : Plants and animal cells, microorganisms, enzymes or their products.

Development of biotechnology in terms of its growth, occurred in two phases viz, Traditional biotechnology and Modern biotechnology.

Traditional or old biotechnology: Based on fermentation technology using microorganisms as in the preparation of curd, ghee, soma, vinegar, yogurt, cheese making, wine making, etc.

Modern or new biotechnology :

There are two major features of technology that differentiate modern biotechnology from classical or old biotechnology

These are,

  • The use of r-DNA technology, polymerase chain reaction (PCR), microarrays, cell culture, cell fusion and bioprocessing to develop specific products.
  • Ownership of technology and its socio political impact.

Principles and Processes of Biotechnology :

Modern biotechnology is based on two core techniques viz. genetic engineering and chemical engineering.

Two core techniques of Biotechnology :

(i) Genetic engineering : Manipulation of genetic material towards a desired end and in a directed and predetermined way, using in vitro process.

  • Definition of genetic engineering (By Smith) : ‘The formation of new combination of heritable material by the insertion of nucleic acid molecule produced by whatever means outside the cells, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host organism in which they do not occur naturally but in which they are capable of continued propagation’.
  • Genetic engineering is also called recombinant DNA technology or gene cloning, as it involves alterations in DNA.

(ii) Chemical engineering: Maintaining sterile environment for manufacturing of useful products like vaccines, antibodies, enzymes, organic acids, vitamins, therapeutics, etc.

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Technique of gene cloning and rDNA technology :

In gene cloning, a gene of known function can be transferred from its normal location into a cell (that of course does not contain it) via a suitable vector. The transferred gene is replicated normally and is handed over to the next progeny.

Tools and techniques for gene cloning/rDNA technology :

(i) The techniques used in r-DNA technology, on the basis of molecular weight : Gel permeation, osmotic pressure, ion exchange chromatography, spectroscopy, mass spectrometry, electrophoresis, etc.

(ii) Electrophoresis :

  • It is used for the separation of charged molecules like DNA, RNA and proteins, by application of an electric field.
  • Different types of electrophoresis : Agarose gel electrophoresis, PAGE, SDA PAGE.

(iii) Polymerase chain reaction (PCR) :

It is used for in vitro gene cloning or gene multiplication to produce a billion copies of the desired segment of DNA or RNA, with high accuracy and specificity, in few hours.

The basic requirements of PCR technique are as follows :

  • DNA containing the desired segment to be amplified.
  • Excess of forward and reverse primers which are synthetic oligonucleotides of 17 to 30 nucleotide. They are complementary to the sequences present in DNA.
  • dNTPs which are of four types such as dATP, dGTP, dTTP and dCTR
  • A thermostable DNA polymerase (e.g. Taq DNA polymerase enzyme) that can withstand a high temperature of 90-98°C.
  • Appropriate quantities of Mg++ ions.
  • Thermal cycler, a device required to carry out PCR reactions.

Three essential steps in PCR :

Three essential steps in PCR : Denaturation, annealing of primer and extension of primer.

  • The DNA segment and excess of two primer molecules, four types of dNTPs, the thermo stable DNA polymerase are mixed together in ‘eppendorf tube’.

One PCR cycle is of 3-4 minutes duration and it involves following steps :

  • Denaturation : The reaction mixture is heated at 90—98°C. Due to this hydrogen bonds in the DNA break and two strands of DNA separate. This is called denaturation.
  • Annealing of primer : When the reaction mixture is cooled to 40—60°C, the primer pairs with its complementary sequences in ssDNA. This is called annealing.
  • Extension of primer : In this step, the temperature is increased to 70-75°C. At this temperature thermostable Taq DNA polymerase adds nucleotides to 3’end of primer using single-stranded DNA as template. This is called primer extension. Duration of this step is about two minutes.
  • In an automatic thermal cycler, the above three steps are automatically repeated
  • 20-30 times.
  • Thus, at the end of ‘n’ cycles 2n copies of DNA segments, get synthesized.

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Biological tools for gene cloning/r-DNA technology :

(1) Enzymes :

  • Lysozymes, Nucleases (exonucleases, endonucleases, restriction endonucleases),
  • DNA ligases, DNA polymerases, alkaline phosphatases, reverse transcriptases, etc.
  • Nucleases : They cut the phosphodlester bonds of polynucleotide chains.

Types of nucleases :

  • Exonucleases : They cut nucleotides from the ends of DNA strands.
  • Endonucleases : They cut DNA from within.

Restriction endonucleases or restriction enzymes :

  • Restriction cutting may result in DNA fragments with blunt ends or cohesive or sticky ends or staggered ends (having short, single stranded projections).
  • Restriction endonucleases have the ability to recognize specific sequences in DNA and cleave it.
  • They are 4 to 8 nucleotides long and characterized by a particular type of internal symmetry.
  • The specific site at which restriction endonuclease cuts the DNA is called recognition site or restriction site.
  • Each restriction endonuclease recognizes its specific recognition sequence.
  • For example, recognition sequence of by the enzyme EcoRI is

3’ ----C T T A A G -----5’

5' ----G A A T T C -----3’

  • It is as palindrome. i.e. when read on opposite strand of DNA (3’ to 5’ or 5’ to 3’) it  reads same.
  • When the enzyme EcoRI recognizes this sequence, it breaks each strand at the same site in the sequence i.e. between the A and G residues.
  • Restriction cutting may result in DNA fragments with blunt ends or cohesive or sticky ends or staggered ends [having short, single stranded projections).

Types of restriction enzyme :

  • Type I : They function simultaneously as endonuclease and methylase e.g. EcoKI.
  • Type II : They have separate cleaving and methylation activities e.g. EcoRI, BgII. They cut DNA at specific sites within the palindrome.
  • Type III : They cut DNA at specific non-palindromic sequences e.g. HpaI, MboII.

(2) Cloning vectors (vehicle DNA) :

  • Vectors are DNA molecules that carry a foreign DNA segment and replicate inside the host cell.

Examples of vectors : Plasmids (e.g. T1 plasmid of Agrobacterium tumefaciens, pBR 322, pUC), bacteriophages (e.g.M13, lambda virus), cosmid, phagemids, BAC (bacterial artificial chromosome), YAC (yeast artificial chromosome], transposons, baculoviruses and MACS (mammalian artificial chromosomes).

Plasmids as cloning vectors :

Plasmids as cloning vectors :

  • Plasmids are small, extra-chromosomal, double stranded circular forms of DNA that replicate autonomously. They are seen in bacterial cells, yeast and animal cell.
  • Plasmids are considered as replicons as they are capable of autonomous replication in suitable host.
  • The most commonly used vectors in r-DNA technology are plasmids as they replicate in E. coli.
  • Plasmid as a cloning vector should have a replication origin, a marker gene for antibiotic resistance, control elements like promoter, operator, ribosome binding site, etc. and a region where foreign DNA can be inserted. Naturally plasmids do not have all these features. Hence, they are constructed by inserting gene for antibiotic resistance. pBR 322, pBR320, paCYC177 are the constructed plasmids.
  • T1 plasmid (for tumor-inducing) of Agrobacterium tumefaciens is an important vector for carrying new DNA in many plants. It contains a transposon, called T DNA, which inserts copies of itself into the chromosomes of infected plant cells. The transposon, with the new DNA, can be inserted into the host cell’s chromosomes. A plant cell containing this DNA, can then be grown in culture or induced to form a new, transgenic plant.

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(3) Competent host :

  • Cloning organism used are usually the bacteria like Bacillus haemophilus, Helicobacter pyroli and E.coli.
  • Mostly E. coli is used for the transformation with recombinant DNA

 Methodology for rDNA technology :

The steps involved in gene cloning :

The steps involved in gene cloning are as follows :

  • Isolation of DNA (gene) from the donor organism.
  • Insertion of desired foreign gene into a cloning vector (vehicle DNA).
  • Transfer of r-DNA into suitable competent host or cloning organism.
  • Selection of the transformed host cell.
  • Multiplication of transformed host cell.
  • Expression of the gene to obtain desired product.

(i) Isolation of DNA (gene) from the donor organism :

  • The desire gene to be cloned has to be obtained from the source organism (donor).The cells of the donor organism are sheared with the blender and treated with suitable detergent. Genetic material from the donor is removed isolated and purified by using several techniques. Isolated DNA can be spooled on to a glass rod.
  • Isolated purified DNA is then cleaved using restriction Endonucleases.
  • Restriction fragment containing desired gene is isolated and selected for cloning. This is now called foreign DNA or passenger DNA.
  • A desired gene can also be obtained directly from genomic library or c-DNA library.

(ii) Insertion of desired foreign gene into a cloning vector (vehicle DNA) :

  • The foreign DNA or passanger DNA is inserted into a cloning vector (vehicle DNA) like bacterial plasmids and the bacteriophages like lamda phage and M13. The most commonly used plasmid is pBR 322.
  • Plasmids are isolated from the bacteria and are cleaved by using same RE which is used in the isolation of the desired gene from the donor.
  • Enzyme DNA ligase is used to join foreign DNA and the plasmid DNA.
  • Plasmid DNA containing foreign DNA is called recombinant DNA (r-DNA) or chimeric DNA.

(iii) Transfer of r-DNA into suitable competent host or cloning organism :

  • The r-DNA is introduced into a competent host cell, which is mostly a bacterium.
  • Host cell takes up naked r-DNA by process of ‘transformation’ and incorporates it into its own chromosomal DNA which finally expresses the trait controlled by passenger DNA.
  • The transfer of r-DNA into a bacterial cell is  assisted by divalent Ca++
  • The cloning organisms are E.coli and  Agrobacterium tumifaciens.
  • The competent host cells which have taken up r-DNA are called transformed cells.
  • By using techniques like electroporation, microinjection, lipofection, shot gun, ultrasonification, biolistic method, etc.
  • Foreign DNA can also be transferred directly into the naked cell or protoplast of the competent host cell, without using vector.
  • In plant biotechnology the transformation is through Ti plasmids of A.tumifaciens.

(iv) Selection of the transformed host cell :

  • For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid vector is employed.
  • For example, pBR322 plasmid vector contains different marker genes like ampicillin resistant gene and tetracycline resistant gene. When pst1 RE is used. It knocks out ampicillin resistant gene from the plasmid, so that the recombinant cells become sensitive to ampicillin.

(v) Multiplication of transformed host cell :

  • The transformed host cells are introduced into fresh culture media where they divide.
  • The recombinant DNA carried by them also multiplies.

(vi) Expression of gene to obtain desired product :

  • Then desired products like enzymes, antibiotiocs etc. separated and purified through downstream processing using bioreactors.

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