BIOTECHNOLOGY: PRINCIPLES AND PROCESSES.
“Biotechnology deals with the techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.” For example- making curd, bread or wine are produced by the action of microbes, could also be considered as a form of biotechnology. But in modern sense it is mainly referred to those processes & products in which genetically modified organisms are used.
The European Federation of Biotechnology (EFB) has given definition of biotechnology by combining traditional and modern view. The definition given by EFB is as.
“The integration of natural science and organisms, cells, parts there of and molecular analogues for products and services.”
Principles of Biotechnology:
The following two core techniques that enabled the birth of modern biotechnology are.
i. Genetic Engineering:
Technique to change the chemistry of genetic material (DNA and RNA), to introduce these into hosts organisms and so change the phenotype of the host.
ii. Maintenance of sterile conditions during genetic engineering so that only desired microbes or eukaryotic cells can grow for manufacture of products like antibiotics, Vaccines, Enzymes etc.
Development of Principles of Genetic Engineering:
Hybridization is the traditional method used in plant breeding and animal breeding for the improvement of the products. Very often it leads to the combination of undesired genes with desired genes. Recombinant DNA can be developed by genetic engineering to over come the limitation of hybridization.
Recombinant DNA:- The host DNA combined with foreign DNA or artificial DNA is called as recombinant DNA.
Origin of replication:- The sequences in DNA which are essential for the initiation of Replication are called as the origin of replication.
The alien DNA is linked with the origin of replication so the DNA can replicate and multiply in the host organism. This is called as DNA cloning.
Construction of 1st Artificial recombinant DNA:
This was done by Stanley Cohen and Herbert Boyer in 1972.
* Plasmids are extra chromosomal DNA which can transfer the genes or DNA one bacteria to another.
* They isolated the antibiotic resistance gene from the plasmid of Salmonella typhimuruim by cutting with restriction enzymes.
* The isolated gene was linked to the plasmid by DNA ligase enzyme. This makes the recombinant DNA in vitro (i.e. in laboratory conditions).
* This recombinant DNA was transferred to the bacteria Escherichia Coli closely related to Salmonella.
* The antibiotic resistant gene can replicate in the E-Coli and multiple copies are formed. This is called as gene cloning.
Three basis steps in Genetically modifying an organism:
i. Identification of DNA with desired genes.
ii. Introduction of the identified DNA into the host.
iii. Maintaining the introduced DNA in host and transfer of the DNA to its progeny.
Tools of Recombinant DNA Technology:
Following tools are required for recombinant DNA technology.
1. Restriction enzymes:-
Restriction enzymes belong to a class of enzymes called as nucleases. These enzymes can cut the DNA double helix at specific sequences. These are of two type- a. Exonucleases b. Endonucleases.
a. Exonucleases:- These enzymes remove nucleotides from the ends of the DNA.
b. Endonucleases: - These make the cuts at specific position with in the DNA. These enzymes recognize specific sequences called palindromic nucleotides sequences in the DNA.
Palindromic nucleotide sequences:- It is the Sequence of the base pairs that reads same on the strands when orientation of reading is kept the same of the following sequences reads same on the two strand in 5’ - 3’ direction or 3’ - 5’ direction.
5’ - GAATTC - 3’.
3’ - CTTAAG - 5’.
The first restriction endonuclease was Hind (II).
Naming of restriction enzymes:
First letter of the name represents of the genus, second letter comes from species of the prokaryotic cell from which they were isolated and Roman number indicated the order. E.g. EcoRI. It is isolated from Eschechia coli R is strain, I- is order of their extraction. Practice Fig11.1for action of EcoRI NCERT Test book page no.196.
Fig. 11.2. Represent recombinant DNA technology NCERT Test book page no. 197.
Separation and isolation of DNA fragments:
The fragments of DNA obtained by the action of restriction enzymes can be separated by the technique known as gel electrophoresis. Since DNA molecules are negatively charged they can move towards anode under electric field through a medium or matrix. The most common matrix is agarose obtained from sea weeds. DNA fragments will separate according to their size through sieving effect provided by the agarose gel hence the smaller the size the farther it moves.
Visualization of DNA fragments:-
The DNA fragments can be stained with ethidium bromide followed by exposure to UV (light) radiations. The bright orange coloured DNA segment will be formed.
The process of extraction of DNA segments from gel is known as elution.
2. Cloning Vectors:- Cloning vectors are the next important tools to restriction enzymes.
Common cloning vectors are plasmids and bacteriophages following features are required to facilitate cloning into a vector.
i. 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 in the next cells.
ii. Selectable markers:-
In addition to ‘ori’ the vector required a selectable marker which helps in identifying the transformants & non transformants eq. genes encoding resistance to antibiotics such as ampicillin, chloraphenical, tetracycline etc. are used as selectable markers.
Transformation: - It is the process by which a DNA piece is introduced in a host bacterium.
iii. Cloning sites:-
These are the sites at which the foreign DNA is combined. These are generally present in the antibiotic resistant genes.
Practice Fig. 11.4.NCERT Test book page no.199
Selection of recombinants using antibiotic resistance genes is a complex process. There for an alternate method is developed, in this the transforments/ recombinants and non recombinants are identified on the bases of their ability to produce colour in the presence of a substrate. In this a recombinant DNA is inserted in the sequence of the enzyme a-galactosidase. This results in inactivation of the enzyme called as insertional in activation so no blue clours will be given by this type of bacteria. Which indicates the recombination. Non recombinants will give the blue colour.
Vectors for cloning genes in plants and animals:
Some bacteria and viruses can be used as vectors for transforming genes.
For Example- Agrebacterium tumifaciens a pathogen on several dicots deliver a piece of DNA called T- DNA and transform the plant calls into tumor and direct the tumor call to produce the chemical required by pathogen. The Ti- plasmid from this bacterium now can be used to deliver genes of our interest into variety of plants.
In animal retroviruses can deliver their DNA into host calls to transform them into concerned cells. So the retroviruses can be used as vector if the harmful DNA can be replaced by the genes of interest.
3. Competent host:-
DNA being the hydrophilic molecule so to pass it through cell membrane the cells are treated with specific concentration of a divalent cations such as calcium. The recombinant DNA can be forced into such cells by incubating the calls with recombinant DNA on ice followed by placing them briefly at 420c (heat shock) and then putting them back on ice.
The other methods of introduction of alien DNA are microinjection and biolistic or gene gun. In microinjection the DNA is directly injected into the nucleus and in gene gun the cell are bombarded with high velocity micro particles of gold and tungsten coated with DNA.
PROCESSES OF RECOMBINANT DNA TECHNOLOGY:
Recombinant DNA technology involves the following steps- i. Isolation of DNA., ii. Fragmentation of DNA., iii. Isolation of desired DNA fragments., iv.Ligation of DNA fragments into vector., v. Transferring the recombinant DNA into host., vi. Culturing of host cells at large level., vii. Extraction of Desired product.
Let us discuss these steps in some details:-
i. Isolation of Genetic material or DNA:-
This can be done by treating plant and animal cells with enzymes such as lysozyme (bacteria), Cellulase (Plant cell), Chitinase (fungus) RNA can be removed by treatment with ribonuclease. Which proteins can be removed by treatment with protease. DNA is ultimately precipitated and by addition of chilled ethanol.
ii. Cutting of DNA at Specific locations:-
This is done by restriction enzymes. These segments are separated by gel electrophoresis. Vector DNA also treated with same enzymes. The desirable segment of DNA is combined with vector DNA with the ligase enzyme to form recombinant DNA.
3. Amplification of Gene of Interest using PCR:-
PCR stands for polymerase chain Reaction. In this reaction multiple copies of DNA or genes of interest can be synthesized invitro using two sets of primers & DNA polymerase enzyme. In this process 1 billion copies of DNA can be made. Each cycle in polymerase chain Reaction has three steps- i. Denaturation. ii. Primer annealing. iii. Extension of primer.
Practice Fig. 11.6. NCERT Text Book. Page no. 202
A thermo stable DNA polymerase enzyme Taq polymerase is used which is isolated from Thermus aquaticus which remains active at high temperature.
4. Insertion of Recombinant DNA in the host:-
Several methods are used. The recipient cells are made competent to receive the foreign DNA.
5. Obtaining Foreign Gene Products:-
These products are obtained on large scale by using bioreactors where large volumes (100-1000 lips) of culture can be processed. A bioreactor provides the optimum condition for growth (temperature, pH, substrate, salts, vitamins, oxygen. The most commonly used bioreactors are of stirring type which are shown in Fig. 11.7. NCERT Text Book.
A bioreactor has an agitator, an oxygen delivery system, and a form control system, a temperature control system, pH control system and sampling parts so that small volume of the culture can withdrawn periodically.
6. Down stream processing:-
This include Separation, Purification, Quality control testing, Clinical trials of drugs etc.
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Very short Answer:-
1. What is Biotechnology?
2. Expand EFB.
3. Write the definition of biotechnology given by EFB.
4. What is recombinant DNA?
5. What is plasmid?
6. What is gene cloning?
7. What is elution?
8. Define transformation.
9. Expand PCR.
10. What are Recombinant proteins?
Short Answer type:-
11. What are the two core techniques which enabled the birth of modern
12. Explain the formation of 1st Recombinant DNA.
13. Give the three basic steps of genetically modifying an organism.
14. What are restriction enzymes? Explain their type and working.
15. Explain the separation of DNA & isolation of DNA fragments.
16. What are cloning vectors? Give example.
17. Explain PCR with suitable diagram.
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