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Published on: 28/09/2019
Biotechnology: Principles and Processes
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1.
"A very small sample of tissue or even a drop of blood can help determine paternity". Provide a scientific explaination to substantiate the statement.
2.
(i) Why was a bactenum used m the first instance of the construction of an artificial recombinant DNA molecule.
(ii) Name the scientists who accomplished this and how?
3.
What does Taq represent in Taq polymerase?
4.
How bacterial cells are made competent to take up DNA?
5.
Give an example where a disease causing pathogen of animals has been transformed into useful vector for delevering genes to humans.
6.
Write the major steps involved in gene cloning.
7.
What is the principle of PCR?
8.
Describe the naming of the restriction enzymes with an example.
9.
Enumerate the process included under Biotechnology.
10.
Explain the process by which a bacterial cell can be made 'competent'. Why is it essential to make bacterial cells 'competent in recombinant' to take up plasmid/rDNA.
11.
How is the amplification of a gene of interest carried out using polymerase Chain Reaction(PCR)?
12.
Explain the basis on which the gel electrophoresis technique works. Write and two ways the products obtained through this technique can be utilised.
13.
Name and explain the technique used in the separation and isolation of DNA fragments to be used in recombinant DNA technology.
14.
Name and describe the technique that helps in separation and isolation of DNA fragments
1.
The above statement can be substantiated through DNA finger print by southern blot method and by PCR.
DNA from all the cells of an individual shows the same degree of polymorphism.
The polymorphs are heritable. An individual and inherits 50% of the chromosomes from maternal and 50% from the paternal parent.
Small amount of DNA from blood or tissue is taken and amplified by PCR, which can be used in DNA finger printing so as to identify the paternity.
2.
(i) Bactrium has a plasmid in which the desired gene is introduced the gene to be transferred is from a bacterium the anti-biotic resistance gene/the host cell (bacterial cell) is required for gene cloning! bacteria produce restriction endonucleases.
(ii) Herbert Boyer and Stanley Cohen Antibiotic resistant gene was isolated using restriction enzyme and introduced into the plasmid of bacterium Sal onella Typhimurium.
Later the recombinant plasmid was mtroduced into the bactenum E coli so that It could make copies of gene.
3.
Taq represents a bacterium Thermus aquaticus which is the source of enzyme DNA polymerase.
4.
Since, DNA is a hydrophilic molecule, it cannot pass through the cell membranes. In order to force bacteria to take up the plasmid, the bacterial cells must first be made competent to take up DNA. This is done by treating them with a specific concentration of a divalent cation such as calcium, which increases the efficiency with which DNA enters the bacterium through the pores in its cell wall.
Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42 C (heat shock) , and then putting them back on ice. This enable the bacteria to take up the recombinant DNA.
5.
The best example is retroviruses which have ability to transform normal animal cells into cancerous cells. These tools of pathogens are now used as vectors for delevering genes of interest to humans. These pathogens are now converted into vectors so that they do not cause any disease.
6.
The technique of gene cloning involves__
(i) Isolation of desired DNA fragment containing the to be cloned ;
(ii) Isolation of the vector used as cloning vehicle ;
(iii) Incubation of DNA fragments and digested vector in presence of DNA ligase producing recombinant DNA molecules : and
(iv) Introduction of recombinant DNA into the host.
7.
The basic principle of PCR is that the double strauded DNA molecule, when heated to a high temperature, the two strands separate yielding single-stranded DNA molecules. The single- stranded DNA molecules can easily be copied with the help of a DNA polymerase and nucleosides resulting in the duplication of original DNA molecule. By repeating these events, multiple copies of the original DNA molecule can be generated.
8.
The first letter of the name comes from the genus of the bacterium.
The second and third letters come from the name of the species of the prokaryote cell from where it is isolated.
The next letter comes from the strain of the prokaryote.
The Roman Numbers following these four letters indicate the order in which the enzymes were isolated from that strain of the bacterium, e.g. EcoRI is isolated from E. coli, R Y 13.
9.
The processes included under biotechnology are:
(i) Recombinant DNA (rDNA) vaccines.
(ii) Synthesis of a gene and introduction of it into a target cell.
(iii) Gene therapy.
(iv) In vitro fertilisation for production of test-tube babies.
(v) Production of many other biological compounds like hormones, insulin, growth hormone, etc.
10.
A bacterial cell can be made competent by treating it with a specific concentration of a divalent cation such as calcium.
Since, DNA is a hydrophilic molecule it cannot pass through cell membrane.
Hence, to force the bacteria to take up the alien DNA, the cells must be made competent; this increases the efficiency with which the DNA enters the bacterial cell through the pores in the cell wall.
11.
Polymerase Chain Reaction:
Multiple copies of the desired gene or segment of DNA, can be synthesised in vitro using two sets of primers, the oligonucleotides that are complementary to the regions of DNA of the two strands and the enzyme DNA polymerase.
This enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as the template.
For repeated amplification to be achieved, a thermostable DNA polymerase (Taq polymerase), extracted from the bacterium, Thermus aquaticus is employed; it remains active during the high temperature treatment used for denaturation and separation of the two strands.
12.
(i) The DNA fragments may be used to construct recombinant DNA by joining them with cloning vectors .
(ii) The desired DNA fragments may be amplified, i.e. making multiple copies, by polymerase chain reaction (PCR).
13.
1. The DNA fragments are separated by electrophoresis using agarose as the matrix.
2. Since, DNA fragments are negatively charged; they move towards the anode under the electric fields through the medium and separate/resolve according to their size due to the sieving effect of agarose gel.
3. The separated fragments can be viewed by staining the DNA with ethidium bromide followed by exposure to UV radiation.
Elution is the process in which the separated bands of DNA are cut out from the gel and extracted.
14.
The DNA fragments are separated by electrophoresis using agarose as the matrix.
Since, DNA fragments are negatively charged; they move towards the anode under the electric fields through the medium and separate/resolve according to their size due to the sieving effect of agarose gel.
The separated fragments can be viewed by staining the DNA with ethidium bromide followed by exposure to UV radiation.
Elution is the process in which the separated bands of DNA are cut out from the gel and extracted.
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