Short & Long Question Answers with Solution: Biotechnology: Principle & Processes | Biology for Grade 12 PDF Download

Short Answer Type Questions

Q1: A mixture of the fragmented DNA was run on an agarose gel. The gel was stained with ethidium bromide but no bands were observed. What would be the cause?
Ans: This could be attributed to the following factors:

• DNA degradation may have occurred due to the action of nucleases.
• The electrodes might have been positioned incorrectly in the gel setup, causing DNA molecules to migrate out of the gel.
• It is possible that ethidium bromide was not adequately incorporated, resulting in the DNA not being visible.

Q2: How is the distinction between recombinants and non-recombinants made using the selectable marker of insertional inactivation of an enzyme?
Ans: By inserting the target gene into the coding region of an enzyme like ß-galactosidase, the presence of the target gene activates the enzyme. In the case of recombinants, it renders the ß-galactosidase gene non-functional. Consequently, when these modified or recombinant hosts are cultured with a chromogenic substrate, they are incapable of producing any coloration. ß-galactosidase is utilized as a selectable marker to differentiate between recombinant and non-recombinant organisms.

Q3: How is Biotechnology useful in developing food crops and in agriculture process?
Ans: Biotechnology has a wide range of applications in the realms of agriculture and the development of food crops.
In agriculture, it plays a crucial role in enhancing the quality, quantity, and processing of food. Eco-friendly solutions like bio-fertilizers and bio-pesticides, which contain living microorganisms, are employed to boost crop growth by augmenting the availability of essential nutrients. Farmers opt for biotech crops to raise yields and reduce production expenses.

Q4: What would happen if a plasmid without a selectable marker was chosen as a cloning vector?
Ans: A selectable marker is essential for distinguishing between transformed cells and non-transformed ones. Without a selectable marker in the cloning vector, identifying the transformants would be a challenging task.

Q5: How does a DNA strand get sticky ends? Why do they go by that name?
Ans: Restriction endonucleases are enzymes responsible for producing sticky ends in DNA. These enzymes cleave the DNA strand at a point between two identical bases on both strands, located slightly away from the center of the palindrome sequence. As a result of this process, both complementary DNA strands possess single-stranded extensions at their ends. These extensions are termed sticky ends because they can form hydrogen bonds with matching base pair sequences.

Q6: What is Biotechnology?
Ans: Biotechnology is a comprehensive field within biology that utilizes technology and the practical application of living organisms and their constituents to create, alter, and produce beneficial products for human well-being. The term 'Biotechnology' was first introduced in 1919 by an agricultural engineer named Karoly Ereky, earning him the title of the father of Biotechnology.

Q7: What would happen if the restriction enzymes do not cut the DNA at specific recognition sequences?
Ans: In cases where restriction enzymes fail to cleave DNA at the designated sites, the resulting DNA fragment will lack sticky ends. Consequently, the process of creating recombinant DNA would become challenging.

Q8: Give a good example to illustrate how to name a restriction endonuclease.
Ans: Naming conventions for restriction endonucleases involve the following rules:

• The first and second letters of the enzyme's name correspond to the genus and species of the prokaryotic cell from which the enzymes are derived.
• Roman numerals following the name indicate the order in which the enzymes were isolated from the bacterial strain. For example, EcoRI originates from Escherichia coli strain RY13, Hind II from Haemophilus influenzae strain Rd, and so on.

Q9: What are the different types of biotechnology?
Ans: The diverse field of Biotechnology has led to a requirement for categorizing it based on shared characteristics or ultimate objectives.
Here are some of the primary domains of Biotechnology:

• Medical Biotechnology.
• Agricultural Biotechnology.
• Industrial Biotechnology.
• Plant Biotechnology.
• Animal Biotechnology.
• Environmental Biotechnology.
• Marine Biotechnology.
• Bio-process engineering.
• Biopharmaceuticals.
• Food Biotechnology.

Q10: Explain the EcoRI mode of action.
Ans: To function effectively, restriction nucleases undergo a process where they assess the length of the DNA sequence, attach themselves to specific recognition sequences, and subsequently cleave the strands at the sugar-phosphate backbones.
There are two distinct categories of these nucleases based on their mode of operation:

• Exonucleases that target the terminal ends of DNA sequences for cleavage.
• Restriction endonucleases, such as EcoRI, which make cuts between the two bases of the recognition sequence.

Long Answer Type Questions

Q1: Mention any three vector-less methods that are used to introduce recombinant DNA into a competent host cell.
Ans: The three vector-less techniques consist of:

• Transformation: This process involves bacteria directly acquiring genetic material from their environment. It requires treating bacterial cells with calcium chloride, incubating them on ice, and subjecting them to high temperatures. This leads to the creation of pores in the bacterial cell wall through which foreign DNA can enter.
• Microinjection: In this method, recombinant DNA is injected directly into the nucleus of an animal cell using a microneedle.
• Biolistics/Gene Gun Method: Cells are bombarded with high-velocity microparticles consisting of gold and tungsten-coated DNA.

Q2: Describe the functions of (i) high temperature, (ii) primers, and (iii) bacteria.
Ans: Polymerase chain reaction (PCR) relies on the activity of an enzyme called Taq polymerase, which is obtained from the bacterium Thermus aquaticus.

• Heat Effect: PCR involves heating the DNA strands to 95°C for two minutes, causing the hydrogen bonds between the bases to break and the strands to separate.
• Primer Function: PCR requires the use of short DNA segments, known as primers, which are approximately 20 base pairs long. These primers play a crucial role in initiating DNA polymerization. They hybridize to their complementary sequences on the DNA strands at a temperature ranging from 40–50°C, facilitating DNA synthesis.
• Role of Thermus aquaticus: Thermus aquaticus is the source of Taq polymerase, an enzyme used in PCR. This bacterium can thrive at high temperatures, up to 95°C, and its Taq polymerase enzyme is heat-resistant, making it suitable for use in PCR where high-temperature conditions are required to denature DNA.

Q3: What are the properties of a good vector?
Ans: An ideal vector should exhibit the following characteristics:

• Compact Size: The vector's size should be minimized for straightforward isolation and purification.
• Replication Origin: It must contain an origin of replication, a specific DNA sequence where the replication process initiates.
• Selectable Marker: The vector should incorporate a selectable marker, aiding in the identification and isolation of the transformed host cells.
• Unique Recognition Site: At least one distinct recognition site should be present on the vector to facilitate the binding of foreign DNA.

Q4: Why were bacteria used to create a synthetic recombinant DNA molecule for the first time? Mention the scientists who did this and how they did it.
Ans: Salmonella typhimurium was selected as the initial artificial recombinant DNA molecule due to the feasibility of combining an antibiotic resistance gene with a native plasmid of this bacterium. This achievement was facilitated by the availability of DNA ligase and restriction enzymes.
In 1972, Stanley Cohen and Herbert Boyer managed to extract the antibiotic resistance gene by excising a specific DNA segment from a plasmid. The advent of restriction enzymes allowed for precise DNA cutting at defined locations. Subsequently, the cut DNA fragment was fused with the plasmid DNA using the DNA ligase enzyme. This DNA attached to the plasmid acted as a vector for transfer.

Q5: What is a polymerase chain reaction? What are the steps involved? Mention its applications.
Ans: Polymerase chain reaction (PCR) is a molecular biology technique employed to replicate numerous copies of a particular DNA segment.

The procedure encompasses the subsequent phases:

• Denaturation
• Annealing
• Extension

PCR is put to use in various domains:

• Forensic Science
• Genetic Research
• Medical Applications

Q6: What function(s) do each of the following play in biotechnology? 

• Gel-electrophoresis
• Restriction endonuclease 
• pBR322’s selectable markers

Ans: 

• Gel-electrophoresis is a technique used for separating and visualizing DNA fragments within an agarose gel matrix. Since DNA fragments are negatively charged, they migrate towards the anode (+ ve) under the influence of an electric field. The gel matrix acts as a sieve, sorting the DNA fragments based on their sizes.
• Restriction endonucleases, or simply restriction enzymes, are bacterial enzymes that recognize specific nucleotide sequences, known as "recognition sites," on double-stranded DNA (dsDNA) and cleave it into fragments. These enzymes are essential for creating recombinant DNA molecules containing DNA from various sources.
• In the case of pBR322, a widely used E. coli cloning vector, selectable markers play a crucial role in identifying transformants. This vector carries two antibiotic resistance genes, ampicillin and tetracycline, which serve as selectable markers. When foreign DNA is ligated into the tetracycline resistance (tetR) gene site of pBR322, the insertional inactivation of tetR results in the loss of tetracycline resistance. However, transformants can still be distinguished from non-recombinants by growing them on ampicillin-containing medium. Recombinant organisms thrive on ampicillin medium but not on tetracycline medium, while non-recombinant organisms grow on both media.

Q7: Give an explanation for the following statements. 

• DNA cannot cross a cell membrane into a host cell. 
• When DNA is isolated for genetic engineering, proteases are added. 
• In a vector, a single cloning site is recommended.

Ans: 

• The lipid bilayer of the plasma membrane is impermeable to hydrophilic molecules, allowing only hydrophobic molecules to diffuse through. DNA's sugar-phosphate backbone, being hydrophilic, cannot pass through the cell membrane.
• Proteases are enzymes that break down proteins into individual amino acids when present in a solution. Failure to remove these proteins during the DNA preparation process, such as through the action of restriction endonuclease and DNA ligase, may lead to interference with subsequent DNA treatments.
• Single cloning sites are preferable over multiple recognition sites within a vector because the latter can generate fragments, complicating the gene cloning process.

Q8: How does the selectable marker function of the p-galactosidase coding sequence? Why is it preferred to antibiotic resistance genes as a selectable marker? Explain.
Ans: A selectable marker is a feature that aids in identifying or selecting transformants while eliminating non-transformants and allowing the selective growth of transformants.
ß-galactosidase serves as a selective marker by causing transformed cells to undergo insertional inactivation. In this process, recombinants and non-recombinants are distinguished based on the amount of color produced when a chromogenic substrate is present. The activity of the enzyme ß-galactosidase is disrupted when recombinant DNA is inserted into its coding region. Consequently, bacterial colonies containing implanted plasmids do not exhibit coloration, whereas those without plasmids do not show color.

Q9: Why and how is it possible to “compete” bacteria?
Ans: DNA molecules cannot naturally pass through cell membranes because they are hydrophilic. To introduce recombinant DNA into cells and have it integrated into the host genome or vector, host cells must be made competent in biotechnology experiments.
There are two methods for making cells competent to accept DNA:

• Chemical transformation: This method involves treating host cells with a specific concentration of a divalent cation, like calcium, to increase the pore size of the cell membrane. Incubating DNA with these treated bacterial cells at 42°C enhances the ability of DNA to enter the cells through cell wall pores.
• Heat-shock method: In this approach, cells infected with recombinant DNA are first incubated on ice for a brief period, followed by a quick heat treatment to 42°C, and then returned to ice.

Q10: What is the PCR technique? What are the many phases in this approach described?
Ans: Polymerase chain reaction (PCR) is a method used to amplify millions of copies of DNA from a single DNA sample. It employs two primary reagents: primers, short segments of single-stranded DNA with a specific sequence that matches the DNA template, and DNA polymerase. Taq polymerase, derived from the bacterium Thermus aquaticus, is a DNA polymerase that can withstand high temperatures.
Each PCR cycle consists of three steps.

• Denaturation: During the first step, a process called DNA denaturation causes the two strands of the DNA helix to physically separate at elevated temperatures.
• Annealing: In the second step, the temperature is lowered to allow the primers to bind to complementary sequences in the DNA.
• Extension: The third step involves synthesizing copies of the target DNA sequence through primer extension. The temperature is raised to 75°C, at which Taq polymerase initiates DNA synthesis starting at the primer's 3'-OH end.