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Karnataka 2nd PUC Biology Question Bank Chapter 11 Biotechnology: Principles and Processes
2nd PUC Biology Biotechnology: Principles and Processes One Mark Questions and Answers
Do eukaryotic cells have restriction endonucleases? Justify your answer.
The eukaryotic cells do not have restriction endonucleases. The eukaryotic cells have some other means of defence i.e., immune system) against viral infection.
Which is the commonly used host cells in genetic engineering?
Which are the common vectors used for cloning genes in plants?
Tiplasmid present in Agrobacterium tumefaciens.
Who discovered the technique of PCR?
What is a bioreactor?
It is an engineering setup designed to carry out biological reactions under aseptic conditions.
Name the technique by which DNA fragments can be separated?
Name the source of Taq polymerase?
What are recombinant proteins?
Proteins produced from a recombinant DNA or transgenic organism are recombinant proteins.
Name the commonly used vector for transformation in plant cell?
Who isolated Restriction enzyme for the first time?
Warner Arber and Hamilton Smith.
Define a patent?
Patent is the govemement protection to the inventor of biological material, securing to him for a specific time the exclusive right of manufacturing exploiting, using and selling an invention.
Define the term plasmid.
Autonomously replicating circular extra chromosomal DNA (or) A circular double stranded, extra chromosomal DNA, present in the cytoplasm of bacteria.
The integration of natural science and organisms, cells, parts thereof and molecular analogues for products and services is known as Biotechnology.
Name the compound used for visualizing DNA under UV radiation.
2nd PUC Biology Biotechnology: Principles and Processes Two Mark Questions and Answers
Describe briefly the following:
(a) Origin of replication
(c) Downstream processing.
(a) Origin of Replication: This is the sequence in DNA molecule, from where, replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cell.
(b) Bioreactors: These are culture vessels in which large volume (100-1000 liters) of culture can be processed to get large quantity of a product on a commercial scale.
(c) Downstream Processing: After the formation, a product, undergoes through some processes before a finished product is ready for marketing. These processes include separation and purification which are collectively called as downstream processing.
(b) Restriction enzymes and DNA
(a) PCR: PCR stands for Polymerase Chain Reaction. In this reaction multiple copies of the gene (or DNA) of interest is synthesised vitro in, using sets of primers and the enzyme Taq DNA polymerase.
(b) Restriction Enzymes and DNA: These are the enzymes which restrict the growth of bacteriophages. These enzymes are present in many bacteria where they function as a part of their defence mechanism called Restriction Modification System. Restriction enzymes are of two types :
(a) Restriction endonuclease cuts DNA into pieces.
(b) Modification enzyme adds methyl group to DNA.
(c) Chitinase: It is an enzyme which breaks down the chitinous substance present in the wall of fungi.
Discuss with your teacher and find out how to distinguish between the following:
(a) Plasmid DNA and Chromosomal DNA
(b) RNA and DNA
(c) Exonuclease and Endonuclease.
(a) Plasmid DNA and Chromosomal DNA: Plasmid DNA is a small double stranded circular DNA present in some bacteria. They carry genes for drug resistance, N2 fixation and fertility. The chromosomal DNA is much larger in size and carries genes for other traits of the cell.
(b) RNA and DNA: RNA is a single stranded polymer of ribonucleotides, which contain ribose sugar and adenine, guanine cytosine and uracil nitrogenous bases, whereas DNA is a double stranded polymer of deoxyribo-nucleotides containing deoxyribose sugar and adenine, guanine, cytosine and thymine nitrogenous bases.
(c) Exonuclease and Endonuclease: Exonucleases remove nucleotides from the ends of DNA, whereas endonucleases make cuts at specific positions within the DNA.
Mention the tools used in recombinant DNA technology.
Desired gene, vector DNA, enzymes, host cell and Bioreactor.
What are palindromic sequences? Give an example.
In a double stranded DNA molecule, if the two strands in a region are identical when read both in forward and backward direction, they are referred to as palindromic sequences.
How are restriction endonuclease named?
The naming of restriction endonuclease is based on the following rules :
- The first letter of the REN refers to the genus name of the bacteria from which it is derived and written in capitals.
- The second and the third letters refer to the species name of the bacteria from which it is derived and is written as a small letter.
- If a fourth letter is present it represents the name of the bacterial strain or subspecies.
- The Roman number indicates the different REN’s derived from the same organism.
Eg.: Hind III.
What are molecular scissors? Explain their role.
The nuclear enzymes that break DNA at specific sites are called restriction endonucleases [REN]. The RENs are popularly known as Biomolecular scissors. REN may cut both the strands of a DNA at the same position or at different positions,
e.g.: Eco RI, Hind III, Sal I, Bam I.
Draw a neat labelled diagram of plasmid pBR322
Write the applications of PCR technique.
- To amplify DNA and RNA strands.
- To study the orientation and location of restriction fragments relative to one another.
- Detection of mutations/presence of mutated genes in the chromosomes.
- To identify DNA finger prints.
What are nucleases? Distinguish between exonucleases and endonucleases.
Restriction enzymes are the nucleases. They are of two types namely exonucleases and
Exonucleases remove nucleotides from the ends of the DNA and endonucleases can cut at specific points within the DNA itself
Draw a neat labeled diagram of stirred tank bio-reactor.
What are ‘Selectable markers”? What is their use in genetic engineering?
A selectable markers is a gene which helps in selecting those host cells which contain the vector and eliminating the non-transformants, e.g. gene encoding resistance to antibiotics are useful selectable markers as they allow selective growth of transformants only.
What is “Insertional inactivation”?
If a recombinant DNA is inserted within the coding sequence of enzyme B – galactosidase, it results in the inactivation of the enzyme which is referred to as “Insertional inactivation”. The presence of chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert presence of insert results in insertional inactivation and the colonies do not produce any colour.
Draw a neat labelled diagram of sparged stirred tank bioreactor.
What are bioreactors? Name the most commonly used bioreactor in genetic engineering.
It is an engineering setup designed to carry out biological reactions under aseptic conditions.
2nd PUC Biology Biotechnology: Principles and Processes Three Marks Questions and Answers
What are the two basic techniques involved in modern Biotechnology?
The two basic techniques involved in modem Biotechnology are
(a) Genetic Engineering which is the technique of altering the nature of genetic material or introduction of it into another host organism to change its phenotype.
(b) Techniques to facilitate the growth and multiplication of only the desired microbes or cells in large number under sterile conditions for manufacture.
What are genetically modified organisms? Name two factors on which their behaviour depends?
Those organisms whose genes have been altered by manipulation, are called genetically modified organisms or transgenic organisms. The two factors on which their behaviour depends are:
- proper insertion of gene of interest.
- proper harvesting of genetically modified organisms to produce the desired product.
What do you mean by “Biopiracy” Give an example?
Biopiracy refers to the use of bio-resources by multinational companies and other oganizations without proper authorizations from the countries and people concerned eg. Basmati rice grown in India, is distinct for its unique flavour and aroma but an American company got patent rights on Basmati through US patent.
2nd PUC Biology Biotechnology: Principles and Processes Five Marks Questions and Answers
Explain the process of recombinant DNA technology.
Process of genetic engineering :
(a) Isolation of genetic material (DNA): In recombinant DNA technology, it is essential to isolate DNA in pure form free from other macro molecules. Since DNA molecule is enclosed with the membrane in the cell, we have to break open the cell to release DNA along with other macromolecules like RNA, proteins, polysaccharides and lipids. This is carried out in bacterial cells, plant and animal cells with certain enzymes.
The other macro molecules can be removed by appropriate treatment with specific enzymes. Finally, the purified DNA molecules are precipitated out after the addition of chilled ethanol and this can be seen as collection of fine threads in the suspension.
(b) Cutting of DNA at specific locations: The isolated purified DNA molecule is cut (cleaved) with the help of a suitable enzyme called restriction endonuclease, into segments with sticky ends.
(c) Gel electrophoresis: The cut DNA fragments are separated by gel electrophoresis using agarose gel. DNA is a negatively charged molecule, hence it moves towards the positive electrode (anode).
(d) Amplification of Gene of interest using PCR: Amplification of gene is ‘a process of making many copies of a gene’. It is achieved by using a technique called Polymerase Chain Reaction (PCR).
Procedure of PCR :
1. The DNA from the desired segment to be amplified, an excess of the two primer molecules, the four deoxyribose triphosphates and DNA polymerase are mixed together in a reaction mixture in a eppendorf tube with sufficient quantities of Mg++. The eppendorf tube is placed in the PCR unit and the following operations are performed sequentially.
2. Denaturation: The reaction mixture is first subjected to a temperature between 90 – 98°C (commonly 94°C). It results in the separation of two strands of DNA due to the breakage of hydrogen bonds. This is called denaturation. Each strand of DNA acts as a template strand for DNA synthesis. The duration of this step in the first cycle of PCR is usually 2 minutes at 94°C.
3. Annealing (anneal=join): The mixture is now cooled to a low temperature (40- 60°C). During this step, two oligonucleotide primers, complementary to a region of DNA, anneal (hybridize) one to each 3 end of DNA strand. The duration of annealing step is usually one minute during the first as well as the subsequent cycles of PCR.
4. Primer extension: During this step, the enzyme taq DNA polymerase extend the primers using nucleotides and DNA templates. The two primers extent towards each other in order to get two new strands of DNA (at 5) end. The duration of primer extension is usually 2 minutes at 72°C. The amplified fragment if required can now be used to ligate with Vector for further cloning. The taq DNA polymerase remains active, during the high temperature induced denaturation of double stranded DNA.
(e) Insertion of recombinant DNA into the host:
1. Electroporation: The bacterial cell is placed in a solution with cold CaCl2 solution followed by placing them at 42°C intermittently. This results in the development of pores in the cell membrane. Now the recombinant plasmid migrates into the host cell and the bacterial cell gets transformed.
2. Microinjection: It is direct injection of a desired gene into the nucleus of an animal cell by microsyringe.
3. Biolistics: Here a suitable plant-cell is bombarded with high velocity microparticles (2-2mm) of gold or tungsten coated with DNA in order to introduce the DNA into the cell.
(f) Obtaining the foreign gene product:
- The transgene expresses itself in the form of protein (s) under appropriate conditions.
- The product (s) can be extracted from the medium by employing a suitable procedure.
- The transgenic cells may be cultured in the laboratory to obtain the transgene product on a small scale.
- The transgenic cells can also be cultured/multiplied in a continuous culture system, in which the used medium is drained out from one side and fresh medium is added from the other side for the production of larger biomass and the desired product.
- Bioreactors are used for processing large volumes of culture for obtaining the product of interest in sufficient quantities on a commercial scale.
(g) Downstream Processing: The products formed in a bioreactor have to be subjected ‘ through a series of processes before they are ready for marketing as finished products.
The various processes used for the recovery of useful products are collectively called downstream processing. The processes include separation and purification of the product, addition of suitable preservatives and a stringent quality control testing etc. Such formulation has to undergo strict clinical trials as in the case of drugs. These quality control testing and clinical trials vary from product to product.
With the help of diagram explain plasmid pBR322.
pBR-322 is a natural plasmid and F+ plasmid. It is about 4.3 kb in size. It is a plasmid with an ori site (origin of replication), two antibiotic resistance sites, selectable markers for Amphicillin resistance (Ampr) and Tetracycline resistance (Tetr). It has thirteen unique sites in different regions out of which seven are important. A unique site is a specific restriction enzyme (REN) recognition site called ECORI.
Vectors for cloning genes in plants and animals are Ti plasmid isolated from Agrobacterium tumefaciens in plants and retrovirus are now made non pathogenic and are used to deliver gene into animal cells.
Note: Insertional inactivation: When a gene or recombinant DNA is inserted within the coding sequence of a vector, the coding sequence responsible for an enzyme or a particular character becomes inactivated. This is known as Insertional inactivation.
a) What is gel electrophoresis? Explain how DNA fragments are separated and detected using this technique.
b) What are plasmids? Mention any two features of an ideal plasmid.
(a) The cutting of DNA by restriction endonucleases results in the fragments of DNA. These fragments can be separated by a technique known as gel electrophoresis. Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix.
Now a days the most commonly used matrix is agarose which is a natural polymer extracted from sea weeds. The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. Hence, the smaller the fragment size, the farther it moves.
The separated DNA fragments can be visualised only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation (you cannot see pure DNA fragments in the visible light and without staining). You can see bright orange coloured bands of DNA in a ethidium bromide stained gel exposed to UV light.
The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution. The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.
(b) The small circular and extra chromosomal double stranded DNA molecules present in the cytoplasm of bacteria are called plasmids. They show self replication. They contain 2 to. 8 thousands of nitrogen base pairs. They also contain some genes, like antibiotic resistant genes and genes for expression of some characters. They can be cut at specific sites using restriction enzymes and desired genes can be inserted into these plasmids.
There are different kinds of plasmids, e.g. F-plasmid [Sex, plasmid], R-plasmid [resistant plasmid], col plasmid [colicins-that kill some strains]. In additions to these there are some genetically engineered plasmids like pBR-322 [Plasmid of Boliver and Rodriguez] and pUC-18 [Plasmid of University of California]
Mention any three tools used in genetic engineering with examples for each.
(a) Vectors eg. plasmid, cosmid.
(b) RENe.g. : Sal I, Bam HI
(c) Host cell eg. E. coli