CBSE Class 12 Biology Revision Notes Chapter 11 - Biotechnology - Principles And Processes
REVISION NOTES FOR CBSE CLASS 12 BIOLOGY CHAPTER- 11
CHAPTER 11: BIOTECHNOLOGY - PRINCIPLES AND PROCESSES
Genetic Engineering (Recombinant DNA Technology)
Insolation of DNA from organism by using enzymes like lysozymes (bacterial cells), Chitinase (fungal cell wall), protease (Proteins), RNA S (RNA) and precipitating in chilled ethanol.
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Cutting of DNA at recognition sites by Restriction enzymes. The same enzyme cuts the cloning vector a similar. recognition sire providing sticky ends.
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The cut fragments are separated using gel electropheresis and amplified using PCR.
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The genes (DNA-fragments) are joined with the cloning vector DNA using ligase.
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The re-combinant DNA so formed is transferred into host cell using methods like biolistics, Electroparation, Micro injection or pathogens. like bacteria and retroviruses whose pathogenic properties have been removed.
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The host cell containing the r-DNA is cultured in Bioreactors to provide the product at large scale.
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The product is separated, purified, (downstream processing) formulated with preservation followed by quality control testing and marketing.
GEL ELECTROPHORESIS
Negatively charged DNA fragments are Separated by forcing them to move through a garose get towards anode under an electricfield.
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The smaller fragments move faster through the gel towards anode
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The larger fragments remain near the walls at the cathode end (where poured initially) as they shieve slowly)
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The separated fragments are stained with ethidium bromide and visualized under UV light
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The DNA fragments are cut out from agarose gel-the process known as elution.
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These DNA fragments are used in recombinant DNA by joining them with cloning vectors
POLYMERASE CHAIN REACTION (PCR)
Denaturtion-Separation of DNA into single strand) by applying high temperature upto 95°C
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Annealing-Two sets of primers (Short stretches of RNA) attach to the single stranded DNA at compementary sites.
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Extension-The primers extend by addition of nucletides in the presence of thermostable DNA polymerase complimentary to the DNA stranu. The primers are removed.
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Repeatation - This cycle get repeated so time and the DNA fragments get amplified about one billion times.
Biotechnology: The application of living organisms or of substances made by living organisms to make products for welfare of mankind.
The definition of Biotechnology given by the European Federation of Biotechnology (EFB): "The integration of natural science and organisms, cells, parts there of, and molecular analogues for products and services.
Molicular scissors- Restriction endonuclease Molicular glues- DNA ligases
Natural genetic engineer- Agrobacterium tumefaciens Three basic steps involved in creating genetically modified organism (GMD) or transgenit organisms
(i) Indentifications of DNA with described genes
(ii) Introduction of the identified DNA into the host
(iii) Maintenance of Introduced DNA into the host and transfer of DNA to its progeny
PRINCIPLES OF BIOTECHNOLOGY:
1. Genetic Engineering: The techniques used to alter the chemistry of genetic material (DNA/RNA) and introduction of it into organisms to change its phenotype.
2. Chemical Engineering: Use of contamination free chemical engineering process of growth of desired microbe or cell in large quantity to obtain bio-technological product like enzyme, antibiotic, vaccine etc.
FIRST ARTIFICIAL RECCOMBINANT DNA MOLECULE :
1. The two scientists of USA, Stanley Cohen and Herbert Boyer (1972) isolated the antibiotic resistance gene by cutting the desired piece of DNA from the plasmid of the bacterium Salmonella typhimurium with the help of restriction enzymes (molecular scissors).
2. This piece of DNA was then linked with the plasmid DNA acting as vector by DNA ligase enzyme.
3. The newly formed recombinant DNA was transferred to bacterium Escherichia coli for replication by using the enzyme DNA polymerase. This process is called Cloning.
Recombinant DNA (rDNA): The hybrid DNA formed by combining DNA segment of two different organisms.
(a) Exonucleases: Cut off nucleotides from terminal ends of DNA
(b) Endonucleases: Make cut DNA at any point within a DNA.
(c) Restriction Endonucleases: Make cut only specific position within a DNA. Single stranded free ends of DNA which can form hydrogen bonds with their complementary cut DNA segments are called "Sticky Ends'. These ends can be joined by enzyme ligase.
(2) Lysing Enzymes: These enzymes are used to open the cells to get DNA. For example: Lysozyme is used to dissolve the bacterial cell wall.
(3) Synthesizing:
(a) Reverse Transcriptases: Used in the synthesis of Complementary DNA strands on RNA templates.
(b) DNA Polymerases Used in the synthesis of Complementary DNA strands on DNA templates.
(4) Joining Enzymes: Are used to join the cut ends of double stranded DNA (act as molecular glue). They join DNA fragments by forming phosphodiester bonds e.g., Ligase.
(5) Alkaline Phosphatases: These enzymes cut the phosphate group from the 5' end of linearised circular DNA to check its recircularization.
SOME RESTRICTION ENZYMES
Palindromic Sequence: Complementary DNA sequences that are the s when each strand is read in the same direction (5'3'). These sequence aci recognition sites for restriction endonuclease.
5'-GAATTC-3'
3'-CTTAAG-5'
Complementary DNA (cDNA): A DNA strand formed from mRNA by using the enzyme reverse transcriptase.
Cloning Vectors: A small, self-replicating DNA molecule into which foreign DNA is inserted. It replicates inside the host cell. The vectors that may be used in genetic engineering are plasmids, bacteriophages, animal, plant, virus, YACS and BACs and some yeasts.
Plasmid : Extra chromosomal, self replicating circular
DNA molecule found in certain bacteria and in some yeasts. It has a few genes. Plasmids are used as cloning vectors in genetic engineering. Plasmids were discovered by Willium Haes and Joshua Leduberg in 1952. The most widely used vector in cloning is pBR322. (an artificial plasmid)
Bacteriophage: A virus which infects bacteria is called bacteriophage.
Features of cloning vector: Origin of replication (Ori), selectable marker and cloning sites are the features that are required to facilitate cloning into a vector.
(a) Origin of Replication (Ori): This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA.
(b) Selectable Marker: It is a gene which helps in identifying and eliminating non-transformants from transformants (having recombinant DNA) by selectively permitting the growth of transformants. The process. through which as piece of DNA is introduced in a host bacterium is called transformation. The genes encoding resistance to antibiotics are considered useful selectable marker for E. coli.
(c) Cloning Sites: A location on a cloning vector into where a foreign gene can be introduced is called recognition site. The vector must have very few (preferably single) recognition sites. The presence of more than one recognition sites within the vector will produce several fragments which will make the process of gene cloning more complicated. Therefore, the foreign DNA is ligated at a restriction site present in one of the two antibiotic resistance gene.
(d) Small Size of Vector: This facilitates the intoduction of DNA into the host easily.
Insertional Inactivation: This method is used to differentiate recombinants from non-recombinants on the basis of ability to produce colour in the presence of a chromogenic substrate. When a rDNA is inserted in the coding sequence of an enzyme. It results in inactivation of the enzyme. This is called insertional inactivation.
Case I: The absence of insert in the plasmid of bacteria: The presence of chromogenic substrate gives blue coloured colonies of bacteria, hence these bacterial colonies are non recombinant.
Case II: The presence of insert in the plasmid of bacteria :
It results insertional inactivation of the B-gatactosidase, therefore bacterial colonies do not give any colour. Hence the bacterial colonies are recombinant.
Steps in Formation of rDNA by action of EcoRI: EcoRI cuts the DNA between bases G and A only → sticky ends of cut DNAs are formed → DNA fragments join at stickly ends by DNA ligase → Recombinant DNA is formed.
RECOMBINANT DNA TECHNOLOGY:
(i) Isolation of Genetic Material (DNA):
¶ DNA can be obtained from the cell by treating with Enzyme like, Lysozyme for bacteria, Cellulase for plant cell, Chitinase for fungus.
¶ Histone protein and RNA can be removed by treating with proteases and ribonuclease respectively.
¶ Purified DNA precipitated by the addition of chilled ethanol, fine threads of DNA are obtained in the suspension.
GEL ELECTROPHORESIS :
(1) DNA fragments are separated by forcing them to move towards anode under an electric field through a medium. Agarose gel is used as medium.
(2) Ethidium bromide is used as stain for DNA.
(3) Then on exposure to UV-light appear as orange coloured bands.
(4) Separated bands of DNA are cut out from agrose gel, this is called elution.
(5) These DNA fragments are used in recombinant DNA by joining them with cloning vectors.
(ii) Cutting of DNA at specific location: The purified DNA is cut by use of restriction enzymes. Agarose gel electrophoresis is used to check the progression of restriction enzymes digestion.
(iii) Amplification of gene of interest using PCR: Amplification is the process of making multiple copies of desired DNA segment invitro. Polymerase chain reaction involves three steps:
(a) Denaturation: The target DNA is heated to high temperature (94°C), resulting the separation of two strands of DNA. Each strand acts as template.
(b) Annealing: Two oligonucleotide primers anneal to each of the single stranded DNA template.
(c) Extension of Primers: DNA polymerase (Taq polymerase) extends the primers using the nucleotides provided in the reactions. Taq polymerase is a heat stable (Thermostable) DNA polymerase which is isolated from thermophilic bacterium named Thermus aquaticus.
(iv) Ligation: The cut out gene of interest from the source of DNA and cut vector with appropriate space, are mixed and ligase enzyme is added. This results recombinant DNA (r-DNA).
(v) Transfer of recombinant DNA into the host: the ligated DNA is introduced into the recipient cell makes itself competent to receive and take up DNA present in the surrounding.
(vi) Obtaining the foreign gene product: The cell containing the foreign gene is cultured on suitable medium and the product can be extracted from the medium.
Bioreactors are used for processing large volume of culture for obtaining products of interest in sufficient quantities. Bioreactor is a large vessel in which raw material is biologically converted into specific product under optimal condition.
(vii) Downstream Processing: The products so obtained undergo a series of processes before putting them in market as a final product. This process includes separation and purification. The products are formulated with suitable preservation and subjected to quality control testing and clinical trials, (in case of drugs).
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