Telangana TSBIE TS Inter 2nd Year Botany Study Material 11th Lesson Biotechnology: Principles and Processes Textbook Questions and Answers.
TS Inter 2nd Year Botany Study Material 11th Lesson Biotechnology: Principles and Processes
Very Short Answer Type Questions
Question 1.
Define biotechnology.
Answer:
- 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.
- 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.
Question 2.
What are molecular scissors? Where are they obtained from?
Answer:
- Molecular scissors are the restrition endonucleases that recognize and cut specific nucleotide sequences of DNA.
- They are usually obtained from Bacteria. For the first time, Herbert Boyer (1969) isolated two restriction enzymes from E.coli.
Question 3.
Name any two artificially restructured plasmids. [May 2014]
Answer:
- pBR 322 (named after Boliver and Rodriguez)
- pUC 19,101 (named after University of California)
Question 4.
What is EcoRI? How does it function?
Answer:
- EcoRI is a restriction enzyme obtained from a bacterium, Escherichia coli.
- This enzyme specifically recognises GAA sites on the DNA and cuts it between G and A.
Question 5.
What are cloning vectors? Give an example.
Answer:
- The DNA used for transforming and multiplying the foreign DNA sequences in a suitable host is called cloning vector.
- Plasmids, Bacteriophages, Cosmids, and artificial chromosomes are commonly used cloning vectors.
Question 6.
What is recombinant DNA?
Answer:
- The hybrid (chimeric)DNA formed by the intergration of a gene of interest within a suitable vector.
- Both source DNA and vector DNA are cut with same restriction enzyme and are joined with DNA ligase to make rDNA.
Question 7.
What is palindromic sequence?
Answer:
- Palindrome sequence: A sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.
- Eg : 5′ – GAATTC – 3′
3′ – CTTAAG – 5′
Question 8.
What is the full form of PCR? How is it useful in biotechnology? [March 2018]
Answer:
- PCR stands for Polymerase Chain Reaction. In this process, multiple copies of a gene are synthesized using a computerized machine called Thermocycler.
- Multiple copies (1 billion) of the gene of interest are synthesized in vitro using two sets of primers and a DNA polymerase (Taq polymerase).
Question 9.
What is downstream processing? [March 2019, May ’17, Mar. ’14]
Answer:
- 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.
- This includes formulation with preservatives, clinical trials (for drugs) and quality control testing etc.
Question 10.
How does one visualize DNA on an agar gel? [March 2020]
Answer:
- 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.
- Bands of DNA in an ethidium bromide stained gel appear in bright in orange colour under UV light, in an instrument called transilluminator.
Question 11.
How can you differentiate between exonucleases and endonucleases?
Answer:
1. Exonucleases :
Nucleases that cut DNA and remove nucleotides from the ends.
2. Endonucleases :
Nucleases that cut specific positions within the DNA.
Short Answer Type Questions
Question 1.
Write short notes on restriction enzymes.
Answer:
Restriction 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.
They are of two types.
- Exonucleases, which remove nucleotides from the ends of DNA.
- Endonucleases, which cut the DNA at specific portions anywhere within its length.
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.
For example, the following sequence reads the same on the two strands in 5′ → 3′ direction as well as 3′ → 5′ direction
5′ – GAATTC – 3′
3′ – CTTAAG – 5′
The restriction enzymes are named as follows.
- 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.
- 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.
eg : EcoRI from Escherichia coli RY13.
Hind 11 from Haemophilus influenza.
Bam H1 from Bacillus amyloliquefaciens.
Question 2.
Give an account of amplification of gene of interest using PCR.
Answer:
PCR stands for Polymerase Chain Reaction.
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.
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.
Question 3.
What is a bio-reactor? Describe briefly the stirring type of bio-reactor.
Answer:
Bioreactors 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.
- It provides all the optimal conditions for achieving the desired product by providing optimal growth conditions like temperature, pH, substrate, salt, vitamins and oxygen.
- The most commonly used bioreactors are of stirring type as shown in figure.
- The stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.
- The stirrer facilitates even mixing and oxygen availability throughout the bioreactor.
- The components of a bioreactor are
a) An agitator system
b) An oxygen delivery system
c) A foam control system
d) A temperature control system
e) pH control system
f) Sampling ports to withdraw cultures periodically
(a) Simple stirred – tank bioreactor;
(b) Sparged stirred-tank bioreactor through which sterile air bubbles are sparged
Question 4.
What are the different methods of insertion of recombinant DNA into the host cell?
Answer:
Methods of insertion of rDNA into the host cell:
- The rDNA can be forced into the competent cells by incubating the cells with recombinant DNA on ice followed by placing them at 42°C and then putting them back on ice.
- 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.
- 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.
- Disarmed pathogens are used as vectors, when they are allowed to infect the cell, they transfer the recombinant DNA into the host.
Long Answer Type Questions
Question 1.
Explain briefly the various processes of recombinant DNA technology. [Mar. 18 17, 14; May 14]
Answer:
Recombinant DNA technology involves several steps in specific sequence such as
a) Isolation of DNA
b) Fragmentation of DNA by restriction endonucleases
c) Isolation of a desired DNA fragment
d) Ligation of the DNA fragment into a vector
e) Transferring the recombinant DNA into the host
f) Culturing the host cells in a medium at large scale
g) Extraction of the desired product.
a) Isolation of DNA :
- 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.
- RNA can be removed by treatment with ribonuclease.
- Proteins can be removed by treatment with protease.
- Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol.
b) Fragmentation of DNA by restriction endonucleases :
Cutting 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.
c) Isolation of a desired DNA fragment :
Is 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.
i) PCR technique requires :
- A DNA template which is a double-stranded DNA that needs to be amplified.
- Primers are small chemically made oligonucleotides of about 10-18 nucleotides that are complementary to a region of template DNA.
- Enzymes used are Taq polymerase (from Thermus aquaticus) and the vent polymerase (from Thermococcus litoralis).
ii) Steps in PCR :
- Denaturation of double-stranded DNA is carried out by applying high temperature of 95°C for 15 seconds. Each separated single-stranded strand acts as a template for DNA synthesis.
- 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.
- Extension is done by DNA polymerase of primers by adding nucleotides complementary to the template provided in the reaction.
- A thermostable DNA polymerase (taq polymerase) is used in the reaction which can tolerate the high temperature of the reaction.
- These steps are repeated many times to obtain several copies of desired DNA.
d) Ligation of the DNA fragment into a vector requires a vector DNA and source DNA.
- These are cut with the same endonuclease to obtain sticky ends.
- Both are then ligated by mixing vector DNA, gene of interest and enzyme DNA ligase to form recombinant DNA.
e) Insertion of recombinant DNA into the host cell :
Organism occurs by several methods, before which the recipient cells are made competent to receive the DNA.
- If recombinant DNA carrying antibiotic resistance (eg., ampjcillin) is transferred into E.coli cells, the host cell is transformed into ampicillin resistant cells
- The ampicillin resistant gene can be called as selectable marker.
- When transformed cells are grown on agar plates containing ampicillin, only transformants will grow and other will die.
f) Culturing the host cells in a medium at a large scale :
It is carried out in appropriate medium at optimal conditions. The DNA gets multiplied and express itself to form desired products.
g) Extraction of desired gene products :
It is carried out by following steps.
- A protein encoded gene expressed in a heterologous host is called recombinant protein.
- Cells having genes of interest can be grown on a small scale or on a large scale.
- In small scale cells are grown on cultures and then expressed protein is extracted and purified by various separation methods.
- 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.
Question 2.
Give a brief account of the tools of recombinant DNA technology. [March 2020, March 2019, May 2017]
Answer:
The tools of recombinant DNA technology
i) Restriction enzymes
ii) Polymerase enzymes
iii) Ligases
iv) Vectors
v) Host organism
i) Restriction enzymes :
Belong to a larger class or enzymes called Nucleases. These are two kinds.
a) Exonucleases :
Exonucleases remove nucleotides from the ends of the DNA.
b) Endonucleases :
Endonucleases 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.
Eg : EcoRI recognises 5′ GAATTC 3′ sites on the DNA and cuts in between G and A.
Naming of Restriction Enzymes :
- 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.
- 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.
Eg: EcoRI from Escherichia coli RY13
Hind II is from Haemophilus influenzae
Bam HI is from Bacillus amyloliquefaciens
ii) Polymerase enzymes :
Thermas aquaticus a bacterium yields DNA polymerase used in biotechnology.
- This enzyme remains active during the high temperature applied during denaturation of double-stranded DNA.
- It extends the primers using the nucleotides provided in the reaction and the genomic DNA as template.
- Repeated amplification is achieved by this enzyme. The amplified fragments, if desired can be used to ligate with a vector for further cloning.
iii) Ligases :
The enzyme DNA ligase joins the complementary ends of the plasmid DNA with that of desired gene by covalent bonding to regenerate a circular hybrid called Recombinant (r) DNA or chimeric DNA.
iv) Vectors :
The DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host called vector.
Vectors used for multiplying the foreign DNA sequence are called cloning vectors. Commonly used vectors are plasmids, bacteriophages, cosmids.
Properties of cloning vector
- It must have low molecular weight.
- It must have unique cleavage site for the activity of restriction enzymes at single point.
- It must be able to replicate inside the host cell after its introduction (through ori gene – origin of replication).
- The vectors requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of transformants.
v) Host organisms :
Competent host for transformation with Recombinant DNA is required as tool.
Intext Question Answers
Question 1.
Do eukaryotic cells have restriction endonucleases? Justify your answer.
Answer:
No, eukaryotic cells do not have restriction endonucleases. This is because the DNA of eukaryotes is highly methylated by a modification enzyme called m&thylase. Methylation protects the DNA from the activity of restriction enzymes. These enzymes are present in prokaryotic cells were they help prevent the invasion of DNA by virus.
Question 2.
Besides better aeration and mixing properties, what other advantages do stirred tank bioreactors have over shake flasks?
Answer:
The shake flask method is used for a small scale production of biotechnological products in a laboratory. On the other hand, stirred tank bioreactors are used for a large scale production of the biotechnology products stirred tank bioreactors have several advantages over shake flasks.
- Small volumes of culture can be taken out from the reactor for sampling or testing.
- It has a foam breaker for regulating the foam.
- It has a control system that regulates the temperature and pH.
Question 3.
Can you recall meiosis and indicate at what stage a recombinant DNA is made?
Answer:
Meiosis is a process that involves the reduction in the amount of genetic material. It is two types, namely meiosis I and meiosis II. During the pachytene stage of prophase I, crossing over of chromosomes takes place where the exchange of segments between non-sister chromatids of homologous chromosomes takes place. This results in the formation of recombinant DNA.
Question 4.
Describe briefly the following :
a) Origin of replication
b) Bioreactors
c) Downstream processing
Answer:
a) Origin of replication :
Origin of replication is defined as the DNA sequence in a genome from where replication initiates. The initiation of replication can be either uni-directional or bi-directional. A protein complex recognizes the ‘on’ site, unwinds the two strands and initiates the copying of the DNA.
b) Bioreactors :
Bioreactors are large vessels used for the large scale production of biotechnology products from raw materials. They provide optimal conditions to obtain the desired product by providing the optimum temperature, pH, vitamin, oxygen, etc. Bioreactors have an oxygen delivery system, a foam control system, a pH, a temperature control system, and a sampling port to obtain a small volume of culture for sampling.
c) Downstream processing :
Downstream processing is a method of separation and purification of foreign gene products after the completion of the biosynthetic stage. The product is subjected to various processes in order to separate and purify the product. After downstream processing, the product is formulated and is passed through various clinical trails for quantity control and other test.
Question 5.
Explain briefly
a) PCR
b) Restriction enzymes and DNA
c) Chitinase
Answer:
a) PCR :
PCR stands for Polymerase Chain Reaction. 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.
b) Restriction enzymes and DNA :
It is also called as molecular scissors. It belongs to class of enzyme called nucleases. It always cut DNA molecules at a particular point by recognizing a specific sequence of six base pairs known as recognition sequence. DNA : DNA stands for Deoxyribo Nucleic Acid. It is a double stranded molecule. It contains deoxyribose sugar. It has the ability to replicate. It is a component of the chromosome.
c) Chitinase :
Chitinase is a class of enzymes used for the degradation of chitin, which forms a major component of the fungal cell wall. Therefore, to isolate the DNA enclosed within the cell membrane of the fungus, enzyme chitinase, is used to break the cell for releasing its genetic material.
Question 6.
Discuss with your teacher and find out how to distinguish between
a) Plasmid DNA and Chromosomal DNA
b) RNA and DNA
c) Exonuclease and Endonuclease
Answer:
a) Plasmid DNA and Chromosomal DNA :
Plasmid DNA | Chromosomal DNA |
Plasmid DNA is an extra-chromosomal DNA molecule in bacteria that is capable of replicating, independent of chromosomal DNA. | Chromosomal DNA is the entire DNA of an organism present inside chromosomes. |
b) RNA and DNA :
RNA | DNA |
1) RNA is single stranded molecule. | 1) DNA is a double stranded molecule. |
2) It contains ribose sugar. | 2) It contains deoxyribose sugar. |
3) The pyramidines in RNA are Adenine and Uracil. | 3) The pyramidines in DNA are Adenine and Thymine. |
4) RNA cannot replicate itself. | 4) DNA molecules have the ability to replicate. |
5) It is a component of the ribosome. | 5) It is a component of the chromosomes. |
c) Exonuclease and Endonuclease :
Exonuclease | Endonuclease |
It is a type of restriction enzyme that removes the nucleotide from 5′ or 3′ ends of the DNA molecule. | It is a type of restriction enzyme that makes a cut within the DNA at a specific site to generate sticky ends. |
Question 7.
What does ‘H’ in’d’ and ‘III refer to in. the enzyme Hind III?
Answer:
In naming restriction enzymes, the first letter comes from the genus Haemophilus (H)
The next two letters comes from the name of the species influenzae (in).
The next letter comes from the strain of the prokaryote (d).
The Roman numbers following these four letters indicate the order in which the enzymes were isolated from that strain of the bacterium.
Question 8.
Restriction enzymes should not have more than one site of action in the cloning site of a vector. Comment.
Answer:
Cloning sites are required to link alien DNA with vector.
- For this, the vector requires single recognition sites forthe commonly used restriction enzymes.
- Presence of more than one restriction sites within the vector will generate several fragments leading to complication to gene cloning.
Question 9.
What does ‘competent’ refer to in ‘competent cells’ used in transformation experiments?
Answer:
Since DNA is a hydrophillic molecule, it cannot pass through 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 pores in its cell wall.
Question 10.
What is the significance of adding proteases at the time of isolation of genetic material (DNA).
Answer:
Proteins can be removed by treating them with proteases at the time of isolation of genetic material (DNA).
Question 11.
While doing a PCR, ‘denaturation’ step is missed. What will be its effect on the process?
Answer:
Annealing and Extensions and amplifing does not takes place.
Question 12.
What modification is done on the Ti plasmid of Agrobacterium tumefaciens to convert it into a cloning vector?
Answer:
Agrobacterium tumefaciens, a pathogen of several dicot plants is able to deliver a piece of DNA known as T-DNA to transform normal plant cells into tumor and directs these tumor cells to produce the chemicals required by the pathogens. The tumor inducing (Ti) plasmids of Agrobacterium tumifaciens has now been modified into a cloning vector such that it is no longer pathogenic to plants but it still able to use the mechanisms to deliver genes of our interest into a variety of plants.
Question 13.
What is meant by gene cloning?
Answer:
The selected fragments of DNA are inserted into a suitable vector to produce a large number of copies of genes. This is called gene cloning.
Question 14.
Decide the ratio between ester bonds and hydrogen bonds that are broken in each palindromic sequence of a DNA when treated with EcoRI during the formation of sticky ends.
Answer:
Equal ratio 2 : 8, i.e 1: 4.