TS Inter 2nd Year Botany Study Material<\/a> 11th Lesson Biotechnology: Principles and Processes Textbook Questions and Answers.<\/p>\nTS Inter 2nd Year Botany Study Material 11th Lesson Biotechnology: Principles and Processes<\/h2>\n
Very Short Answer Type Questions<\/span><\/p>\nQuestion 1.
\nDefine biotechnology.
\nAnswer:<\/p>\n
\n- The European Federation of Biotechnology (EFB) defines Biotechnology as the intergration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.<\/li>\n
- Biotechnology is the science of utilising the properties and uses of microorganisms or to exploit cells and the cell constituents at industrial level for generating useful products essential to life and human welfare.<\/li>\n<\/ol>\n
Question 2.
\nWhat are molecular scissors? Where are they obtained from?
\nAnswer:<\/p>\n
\n- Molecular scissors are the restrition endonucleases that recognize and cut specific nucleotide sequences of DNA.<\/li>\n
- They are usually obtained from Bacteria. For the first time, Herbert Boyer (1969) isolated two restriction enzymes from E.coli.<\/li>\n<\/ol>\n
Question 3.
\nName any two artificially restructured plasmids. [May 2014]
\nAnswer:<\/p>\n
\n- pBR 322 (named after Boliver and Rodriguez)<\/li>\n
- pUC 19,101 (named after University of California)<\/li>\n<\/ol>\n
Question 4.
\nWhat is EcoRI? How does it function?
\nAnswer:<\/p>\n
\n- EcoRI is a restriction enzyme obtained from a bacterium, Escherichia coli.<\/li>\n
- This enzyme specifically recognises GAA sites on the DNA and cuts it between G and A.<\/li>\n<\/ol>\n
Question 5.
\nWhat are cloning vectors? Give an example.
\nAnswer:<\/p>\n
\n- The DNA used for transforming and multiplying the foreign DNA sequences in a suitable host is called cloning vector.<\/li>\n
- Plasmids, Bacteriophages, Cosmids, and artificial chromosomes are commonly used cloning vectors.<\/li>\n<\/ol>\n
<\/p>\n
Question 6.
\nWhat is recombinant DNA?
\nAnswer:<\/p>\n
\n- The hybrid (chimeric)DNA formed by the intergration of a gene of interest within a suitable vector.<\/li>\n
- Both source DNA and vector DNA are cut with same restriction enzyme and are joined with DNA ligase to make rDNA.<\/li>\n<\/ol>\n
Question 7.
\nWhat is palindromic sequence?
\nAnswer:<\/p>\n
\n- Palindrome sequence: A sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.<\/li>\n
- Eg : 5′ – GAATTC – 3′
\n3′ – CTTAAG – 5′<\/li>\n<\/ol>\nQuestion 8.
\nWhat is the full form of PCR? How is it useful in biotechnology? [March 2018]
\nAnswer:<\/p>\n
\n- PCR stands for Polymerase Chain Reaction. In this process, multiple copies of a gene are synthesized using a computerized machine called Thermocycler.<\/li>\n
- Multiple copies (1 billion) of the gene of interest are synthesized in vitro using two sets of primers and a DNA polymerase (Taq polymerase).<\/li>\n<\/ol>\n
Question 9.
\nWhat is downstream processing? [March 2019, May ’17, Mar. ’14]
\nAnswer:<\/p>\n
\n- Downstream processing : Separation and purification of a product that carried out after completion of the biosynthetic stage and before it is ready for marketing as a finished product.<\/li>\n
- This includes formulation with preservatives, clinical trials (for drugs) and quality control testing etc.<\/li>\n<\/ol>\n
Question 10.
\nHow does one visualize DNA on an agar gel? [March 2020]
\nAnswer:<\/p>\n
\n- 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.<\/li>\n
- Bands of DNA in an ethidium bromide stained gel appear in bright in orange colour under UV light, in an instrument called transilluminator.<\/li>\n<\/ol>\n
<\/p>\n
Question 11.
\nHow can you differentiate between exonucleases and endonucleases?
\nAnswer:
\n1. Exonucleases :
\nNucleases that cut DNA and remove nucleotides from the ends.<\/p>\n
2. Endonucleases :
\nNucleases that cut specific positions within the DNA.<\/p>\n
Short Answer Type Questions<\/span><\/p>\nQuestion 1.
\nWrite short notes on restriction enzymes.
\nAnswer:
\nRestriction enzymes or molecular scissors belong to a class of enzymes called nucleases. It always cut DNA molecules at a particular point by recognizing a specific sequence of six base pairs known as recognition sequence.
\nThey are of two types.<\/p>\n
\n- Exonucleases, which remove nucleotides from the ends of DNA.<\/li>\n
- Endonucleases, which cut the DNA at specific portions anywhere within its length.<\/li>\n<\/ol>\n
Each restriction endonuclease recognizes a specific palindromic nucleotide sequence in the DNA Palindrome is a group of letters that forms the same words when read both forward and backward, eg: MALAYALAM. It is a sequence in DNA of base pairs that reads same on the two strands. When orientation of reading is kept same.<\/p>\n
For example, the following sequence reads the same on the two strands in 5′ \u2192 3′ direction as well as 3′ \u2192 5′ direction
\n5′ – GAATTC – 3′
\n3′ – CTTAAG – 5′<\/p>\n
The restriction enzymes are named as follows.<\/p>\n
\n- The first letter of the name comes from the genus and the next two letters from the name of the species of the prokaryotic cell from which it is isolated.<\/li>\n
- The next letter comes from the strain of the prokaryote.<\/li>\n
- The Roman numbers following these four letters indicate the order in which the enzymes were isolated from that strain of the bacterium.
\neg : EcoRI from Escherichia coli RY13.
\nHind 11 from Haemophilus influenza.
\nBam H1 from Bacillus amyloliquefaciens.<\/li>\n<\/ul>\nQuestion 2.
\nGive an account of amplification of gene of interest using PCR.
\nAnswer:
\nPCR stands for Polymerase Chain Reaction.<\/p>\n
In this reaction, multiple copies of the gene (or DNA) of interest are synthesised in vitro using two sets of primers and the enzyme DNA polymerase. The enzyme extends the primers, using the nucleotides provided in the reaction and the genomic DNA as template.<\/p>\n
If the replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times i.e., 1 billion copies are made. Such repeated amplification is achieved by the use of a thermostable DNA polymerase such as Taq polymerase (isolated from a bacterium Thermus aquaticus) which remain active even during the high temperature induced denaturation of double stranded DNA. The amplified fragment, if desired, can now be used to ligate with a vector for further cloning.
\n<\/p>\n
Question 3.
\nWhat is a bio-reactor? Describe briefly the stirring type of bio-reactor.
\nAnswer:
\nBioreactors are large volume (100 – 1000L) vessels in which raw materials are biologically converted into specific products, individual enzymes etc. using microbial plant, animal or human ceils.<\/p>\n
\n- It provides all the optimal conditions for achieving the desired product by providing optimal growth conditions like temperature, pH, substrate, salt, vitamins and oxygen.<\/li>\n
- The most commonly used bioreactors are of stirring type as shown in figure.<\/li>\n
- The stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.<\/li>\n
- The stirrer facilitates even mixing and oxygen availability throughout the bioreactor.<\/li>\n
- The components of a bioreactor are
\na) An agitator system
\nb) An oxygen delivery system
\nc) A foam control system
\nd) A temperature control system
\ne) pH control system
\nf) Sampling ports to withdraw cultures periodically<\/li>\n<\/ol>\n
\n(a) Simple stirred – tank bioreactor;
\n(b) Sparged stirred-tank bioreactor through which sterile air bubbles are sparged<\/p>\n
Question 4.
\nWhat are the different methods of insertion of recombinant DNA into the host cell?
\nAnswer:
\nMethods of insertion of rDNA into the host cell:<\/p>\n
\n- The rDNA can be forced into the competent cells by incubating the cells with recombinant DNA on ice followed by placing them at 42\u00b0C and then putting them back on ice.<\/li>\n
- Microinjection is a method in which the recombinant DNA is directly injected into the nucleus of the animal cell with the help of microneedles or micropipettes.<\/li>\n
- Gene gun or biolistics is a method suitable for plant cells, where cells are bombarded with high velocity microparticles of gold or tungsten coated with DNA.<\/li>\n
- Disarmed pathogens are used as vectors, when they are allowed to infect the cell, they transfer the recombinant DNA into the host.<\/li>\n<\/ul>\n
Long Answer Type Questions<\/span><\/p>\nQuestion 1.
\nExplain briefly the various processes of recombinant DNA technology. [Mar. 18 17, 14; May 14]
\nAnswer:
\nRecombinant DNA technology involves several steps in specific sequence such as
\na) Isolation of DNA
\nb) Fragmentation of DNA by restriction endonucleases
\nc) Isolation of a desired DNA fragment
\nd) Ligation of the DNA fragment into a vector
\ne) Transferring the recombinant DNA into the host
\nf) Culturing the host cells in a medium at large scale
\ng) Extraction of the desired product.<\/p>\n
a) Isolation of DNA :<\/p>\n
\n- DNA is enclosed within the membranes. To release DNA along with other macromolecules such as RNA, proteins, polysaccharides and lipids, bacterial cells \/ – plants or animal tissue are treated with enzymes such as lysozyme (bacteria) cellulose (plant cells), chitinase (fungus) to digest cell wall.<\/li>\n
- RNA can be removed by treatment with ribonuclease.<\/li>\n
- Proteins can be removed by treatment with protease.<\/li>\n
- Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol.<\/li>\n<\/ol>\n
b) Fragmentation of DNA by restriction endonucleases :
\nCutting of DNA at specific locations can be done by using restriction enzymes. The purified DNA is incubated with the specific restriction-enzymes at conditions optimum for the enzyme to act.<\/p>\n
c) Isolation of a desired DNA fragment :
\nIs carried out using agarose gel electrophoresis the DNA is negatively charged, it moves towards the positive electrode or anode and in the process, DNA separates out. The desired DNA fragment is eluted out. Amplification of the gene of interest: Using Polymerase Chain Reaction (PCR) is a reaction in which amplification of specific DNA sequences is carried out in vitro.<\/p>\n
i) PCR technique requires :<\/p>\n
\n- A DNA template which is a double-stranded DNA that needs to be amplified.<\/li>\n
- Primers are small chemically made oligonucleotides of about 10-18 nucleotides that are complementary to a region of template DNA.<\/li>\n
- Enzymes used are Taq polymerase (from Thermus aquaticus) and the vent polymerase (from Thermococcus litoralis).<\/li>\n<\/ol>\n
ii) Steps in PCR :<\/p>\n
\n- Denaturation of double-stranded DNA is carried out by applying high temperature of 95\u00b0C for 15 seconds. Each separated single-stranded strand acts as a template for DNA synthesis.<\/li>\n
- Annaling is carried out in two sets of primers. Which are added and anneal to the 3′ end of each separated strand. Primers act as initiator of replication.<\/li>\n
- Extension is done by DNA polymerase of primers by adding nucleotides complementary to the template provided in the reaction.<\/li>\n
- A thermostable DNA polymerase (taq polymerase) is used in the reaction which can tolerate the high temperature of the reaction.<\/li>\n
- These steps are repeated many times to obtain several copies of desired DNA.<\/li>\n<\/ol>\n
d) Ligation of the DNA fragment into a vector requires a vector DNA and source DNA.<\/p>\n
\n- These are cut with the same endonuclease to obtain sticky ends.<\/li>\n
- Both are then ligated by mixing vector DNA, gene of interest and enzyme DNA ligase to form recombinant DNA.<\/li>\n<\/ol>\n
<\/p>\n
e) Insertion of recombinant DNA into the host cell :
\nOrganism occurs by several methods, before which the recipient cells are made competent to receive the DNA.<\/p>\n
\n- If recombinant DNA carrying antibiotic resistance (eg., ampjcillin) is transferred into E.coli cells, the host cell is transformed into ampicillin resistant cells<\/li>\n
- The ampicillin resistant gene can be called as selectable marker.<\/li>\n
- When transformed cells are grown on agar plates containing ampicillin, only transformants will grow and other will die.<\/li>\n<\/ol>\n
f) Culturing the host cells in a medium at a large scale :
\nIt is carried out in appropriate medium at optimal conditions. The DNA gets multiplied and express itself to form desired products.<\/p>\n
g) Extraction of desired gene products :
\nIt is carried out by following steps.<\/p>\n
\n- A protein encoded gene expressed in a heterologous host is called recombinant protein.<\/li>\n
- Cells having genes of interest can be grown on a small scale or on a large scale.<\/li>\n
- In small scale cells are grown on cultures and then expressed protein is extracted and purified by various separation methods.<\/li>\n
- In large scale cells are grown in a continuous culture system in which fresh medium is added from one side to maintain cells growth phase and the desired protein is collected from the other side.<\/li>\n<\/ol>\n
<\/p>\n
Question 2.
\nGive a brief account of the tools of recombinant DNA technology. [March 2020, March 2019, May 2017]
\nAnswer:
\nThe tools of recombinant DNA technology
\ni) Restriction enzymes
\nii) Polymerase enzymes
\niii) Ligases
\niv) Vectors
\nv) Host organism<\/p>\n
i) Restriction enzymes :
\nBelong to a larger class or enzymes called Nucleases. These are two kinds.
\na) Exonucleases :
\nExonucleases remove nucleotides from the ends of the DNA.<\/p>\n
b) Endonucleases :
\nEndonucleases make cuts at specific positions with in the DNA. Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands.
\nEg : EcoRI recognises 5′ GAATTC 3′ sites on the DNA and cuts in between G and A.
\n<\/p>\n
Naming of Restriction Enzymes :<\/p>\n