RowQ
The Vault
RowQ
The Vault
CBSE Class 12 Biology · 11 questions · 26 marks
Biotechnology is essentially the art of persuading a bacterium to make a molecule it never evolved to make. This chapter sets out the toolkit that makes that possible: restriction enzymes that cut DNA at defined sequences, plasmid and viral vectors that carry a gene into a host, and PCR that amplifies a fragment millions of times over. Once you can list the steps of recombinant DNA technology in order, the applications chapter that follows becomes much easier.
The restriction enzyme EcoRI recognises the sequence 5' GAATTC 3'. Such a sequence, which reads the same on both strands in the 5' → 3' direction, is described as:
Answer
Palindromic is correct — reading the complementary strand in its own 5' → 3' direction gives GAATTC once again. Restriction enzymes cut such sequences at staggered positions on the two strands, which is precisely why single-stranded sticky ends are produced. Repetitive refers to satellite DNA, while degenerate and overlapping describe properties of the genetic code.
Which enzyme is essential in PCR because it survives the repeated high-temperature denaturation steps?
Answer
Taq polymerase is correct — isolated from the thermophilic bacterium Thermus aquaticus, it remains active at the near-boiling temperatures used to separate the two DNA strands in each cycle, so fresh enzyme need not be added every round. An ordinary DNA polymerase would be denatured in the first cycle; ligase joins fragments, reverse transcriptase makes DNA from RNA, and EcoRI cuts DNA.
During gel electrophoresis, DNA fragments move towards the anode because:
Answer
The phosphate groups give DNA a net negative charge is correct — every nucleotide carries a negatively charged phosphate in the backbone, so the whole molecule migrates towards the positive electrode when a field is applied. The agarose acts only as a sieve, retarding larger fragments so that smaller ones travel further, and ethidium bromide is merely a stain that makes the separated bands visible under UV light.
Which of the following is NOT a required feature of a good cloning vector?
Answer
A gene coding for a human hormone is correct as the answer, because that is the foreign gene one might choose to insert, not a property the vector itself must possess. A vector must have an origin of replication so it can multiply in the host, a selectable marker so transformed cells can be picked out from untransformed ones, and unique restriction sites where the foreign DNA can be spliced in.
Assertion (A): Bacterial cells are treated with divalent cations and given a brief heat shock before recombinant DNA is introduced. Reason (R): The bacterial cell wall is normally impermeable to large hydrophilic molecules such as DNA.
Answer
Both A and R are true and R is the correct explanation of A — since DNA cannot cross the wall of a normal bacterial cell, the cell must first be made competent. Treatment with divalent cations such as Ca²⁺ increases the efficiency with which DNA enters through pores in the wall, and incubating the cells on ice, then briefly at about 42 °C, and returning them to ice enables the recombinant DNA to be taken up.
What are sticky ends, and why are they useful in making recombinant DNA?
Answer
When a restriction endonuclease cuts a palindromic sequence, it does not cut both strands at the same point but at staggered positions a little away from the centre. This leaves each fragment with a short single-stranded overhang at its end, and these overhangs are called sticky ends. They are useful because the sticky ends of any two fragments cut by the same restriction enzyme are complementary to one another. When vector DNA and foreign DNA are cut with the same enzyme and mixed, their sticky ends base-pair by hydrogen bonding, holding the two pieces together so that DNA ligase can seal the backbone and produce a stable recombinant molecule.
Explain insertional inactivation and state why it is preferred over selection based on antibiotic resistance markers alone.
Answer
In insertional inactivation, the foreign DNA is deliberately inserted within a marker gene, so that if insertion has succeeded the marker gene is disrupted and its product is not made. A common version uses a gene coding for the enzyme beta-galactosidase. Bacteria are plated on a medium containing a chromogenic substrate: colonies with an intact gene produce the enzyme, break down the substrate, and turn blue, while recombinant colonies, whose gene has been interrupted by the insert, produce no enzyme and remain white. This is preferred because selection with two antibiotic resistance genes requires the tedious process of replica plating — transferring every colony to a second plate containing the antibiotic and comparing the two plates to find the colonies that have lost resistance. Insertional inactivation with a colour marker lets recombinants be identified directly on a single plate simply by looking at colony colour, which is faster and far less laborious.
Why is a stirred-tank bioreactor preferred over a simple flask for producing a recombinant protein on a commercial scale?
Answer
A shake flask can only handle small volumes of culture, and the conditions inside it cannot be controlled precisely. Large-scale production requires continuous, uniform control of temperature, pH, substrate concentration, dissolved oxygen, and vitamins or salts, because the yield of the product depends on all of these being kept at their optimum. A stirred-tank bioreactor is designed for this. Its agitator system mixes the culture evenly and distributes heat throughout the vessel, the oxygen delivery system and foam control maintain aeration, and the temperature and pH control systems with a sampling port allow conditions to be monitored and corrected during the run. This makes it possible to process hundreds of litres of culture reproducibly and obtain a much larger quantity of usable product.
Outline, in sequence, the steps involved in producing a recombinant protein from a cloned gene, naming the enzymes and equipment used at each stage.
Answer
Isolation of the genetic material: the cell is treated with enzymes to break open its wall or membrane — lysozyme for bacteria, cellulase for plant cells, and chitinase for fungi — and then with ribonuclease to remove RNA and protease to remove protein. The purified DNA precipitates as fine threads when chilled ethanol is added. Cutting the DNA: the isolated DNA and the chosen vector are both cut with the same restriction endonuclease, for example EcoRI. Because the enzyme cuts palindromic sequences at staggered positions, both the gene of interest and the opened vector acquire complementary sticky ends. The fragments are checked by agarose gel electrophoresis, where they separate according to size and are visualised as bands under UV light after ethidium bromide staining; the required band is cut out and eluted. Ligation: the gene of interest and the cut vector are mixed so that their sticky ends base-pair, and DNA ligase seals the nicks in the sugar-phosphate backbone, producing a recombinant DNA molecule. Insertion into the host: the host bacterium is first made competent by treatment with divalent cations such as Ca²⁺, followed by incubation on ice, a brief heat shock at about 42 °C, and a return to ice, which allows the recombinant DNA to enter. Alternatives are micro-injection directly into the nucleus of an animal cell, or the biolistic gene gun, which bombards plant cells with gold or tungsten particles coated with DNA. Selection of transformants: recombinant cells are identified using a selectable marker, most conveniently by insertional inactivation, in which recombinant colonies remain white while non-recombinants turn blue on a chromogenic substrate. Culture and harvest: the selected recombinant cells are grown on a large scale in a bioreactor, commonly a stirred-tank type, which maintains optimum temperature, pH, dissolved oxygen, agitation, and substrate supply for maximum yield of the desired protein. Downstream processing: the product is separated from the culture, purified, and formulated with suitable preservatives, then subjected to strict clinical trials and quality control testing before it can be marketed.
Describe the polymerase chain reaction, explaining what happens in each cycle and giving three applications of the technique.
Answer
The polymerase chain reaction is a method for making a very large number of copies of a chosen segment of DNA in vitro. The reaction mixture contains the template DNA, a pair of short chemically synthesised oligonucleotide primers complementary to the two ends of the region to be copied, the four deoxynucleotides, and a thermostable DNA polymerase. Each cycle has three steps. Denaturation: the mixture is heated to a high temperature, around 94 °C, which breaks the hydrogen bonds between the two strands of the template so that they separate. Annealing: the temperature is lowered, allowing each primer to base-pair with its complementary sequence at one end of the target region on the separated strands. Extension: the temperature is raised to the optimum for the polymerase, and the enzyme extends each primer in the 5' → 3' direction using the deoxynucleotides provided, synthesising a new strand along the template. The key to the method is that the polymerase used is Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus. Because it stays active at the near-boiling denaturation temperature, the same enzyme survives every cycle and fresh enzyme need not be added. Since each cycle doubles the number of copies of the target region, the amplification is exponential — about 30 cycles yield over a billion copies from a single starting molecule. The whole process is carried out automatically in a thermal cycler that changes the temperature on a programmed schedule. Applications include: amplifying a gene of interest from a small sample so that there is enough of it for cloning into a vector; diagnosing infection or genetic disorder by detecting very low amounts of a specific DNA sequence, for example screening a suspected HIV patient long before symptoms appear; and forensic work, where DNA from a minute biological sample recovered at a scene is amplified to a quantity sufficient for DNA fingerprinting. It is also used to detect mutations in genes in suspected cancer patients.
A student team in a university laboratory sets out to clone a bacterial gene coding for a heat-tolerant enzyme. They cut both the source DNA and a plasmid vector with the same restriction enzyme, mix the fragments, and treat them with an enzyme that seals the backbone. The mixture is then used to transform bacteria that have been chilled with calcium chloride and briefly warmed. The bacteria are plated on a medium containing a chromogenic substrate, and the team observes a mixture of blue and white colonies. Their vector carries the insertion site inside a gene for beta-galactosidase. (a) Why must the source DNA and the vector be cut with the same restriction enzyme? (b) Name the enzyme that seals the backbone and the treatment that allowed the bacteria to take up the DNA. (c) Which colonies should the team pick, and why? (d) Having obtained the recombinant bacteria, what should they do next to produce a usable quantity of the enzyme?
Answer
(a) A given restriction enzyme cuts only its own specific palindromic sequence, and always at the same staggered positions, so both the gene fragment and the opened vector end up with identical single-stranded overhangs. Only then are the sticky ends of the two pieces complementary, so that they can base-pair with each other and be joined into a stable recombinant molecule. Fragments cut with different enzymes would have mismatched ends and would not anneal. (b) The backbone is sealed by DNA ligase. The bacteria were made competent by treatment with the divalent Ca²⁺ ions of calcium chloride, followed by incubation on ice, a brief heat shock at about 42 °C, and a return to ice, which allows the recombinant DNA to pass into the cell. (c) They should pick the white colonies. The insertion site lies within the beta-galactosidase gene, so in a recombinant the gene is interrupted and no enzyme is produced, the chromogenic substrate is not broken down, and the colony stays white. A blue colony has an intact gene, meaning the vector re-circularised without taking up the insert. This is insertional inactivation, and it lets recombinants be picked out on a single plate. (d) They should culture the selected recombinant bacteria on a large scale in a bioreactor, typically a stirred-tank type, which maintains optimum temperature, pH, dissolved oxygen, agitation, and nutrient supply for maximum yield. The culture is then taken through downstream processing — separation of the product from the cells and medium, purification, and formulation with suitable preservatives — followed by quality control testing before the enzyme can be used or sold.
RowQ generates fresh questions on Biotechnology - Principles and Processes, marks your answers, and explains every step.
Start free