NCERT Solutions for Class 12 Biology Chapter 9: Biotechnology: Principles and Processes

A recombinant protein is any protein-encoding gene product expressed in a heterologous host using recombinant DNA technology. Since the 1980s, many such proteins, manufactured in genetically engineered bacteria, yeast or mammalian cells, have become standard therapeutics:

Conclusion: recombinant DNA technology has made it possible to mass-produce human proteins — from insulin and growth hormone to clot-busting enzymes and vaccines — in genetically engineered micro-organisms, transforming the treatment of diseases that were once difficult or impossible to manage with naturally extracted proteins.

The restriction enzyme EcoRI (from Escherichia coli RY13) is the best-documented example in this chapter and makes an ideal chart:

Conclusion: EcoRI recognises the palindromic sequence GAATTC, cuts both strands between the G and A bases, and produces two DNA fragments with complementary single-stranded AATT sticky ends — the basic mechanism by which restriction enzymes enable the construction of recombinant DNA.

DNA is much bigger in molecular size than an enzyme.

1. A restriction enzyme such as EcoRI recognises and acts on a tiny, fixed 6-base-pair recognition sequence within a DNA molecule. The DNA molecule itself — whether a bacterial plasmid (a few thousand base pairs) or a chromosome (millions to billions of base pairs) — is a very long polymer chain that contains this 6-bp site at just one or a few points along its enormous length.
2. Because the DNA molecule is so much longer than the enzyme’s recognition site, many individual enzyme molecules can simultaneously bind at different, widely separated points along a single DNA molecule — something only possible if the DNA is far larger than any one enzyme molecule.
3. Structurally, an enzyme is a single folded protein (typically tens of thousands of Daltons in molecular weight), whereas DNA is an extremely long, continuous chain polymer of nucleotides, giving it a molecular weight that can run into millions or even billions of Daltons for a full chromosome.

Conclusion: DNA is the much larger molecule — a restriction enzyme is a comparatively small protein that recognises and acts upon only a short, specific sequence within a DNA strand that is itself a vastly longer polymer, which is why a single DNA molecule can carry binding/cutting sites for many enzyme molecules at once.

This is an estimation exercise, best worked out with reasonable assumptions, as NCERT itself suggests consulting your teacher for this calculation. One standard, illustrative approach:

1. Number of DNA molecules per cell. A normal human somatic (diploid) cell has 46 chromosomes, and each chromosome is essentially one very long DNA molecule — so there are 46 DNA molecules per cell (before replication).
2. Moles of DNA per cell. Using Avogadro’s number (6.022 × 10²³ molecules per mole): moles of DNA = 46 ÷ (6.022 × 10²³) ≈ 7.6 × 10⁻²³ mol.
3. Approximate cell volume. Treating a typical human cell as a sphere of diameter ≈10 µm (radius 5 × 10⁻⁴ cm), volume = ⅓πr³ ≈ 5.2 × 10⁻&sup9; cm³ = 5.2 × 10⁻&sup9;³ L (since 1 cm³ = 1 mL = 10⁻³ L, this is ≈ 5.2 × 10⁻&sup9; mL = 5.2 × 10⁻&sup9;² L — approximately 5 × 10⁻&sup9; mL, i.e., about half a picolitre).
4. Molar concentration. Concentration = moles ÷ volume ≈ (7.6 × 10⁻²³ mol) ÷ (5 × 10⁻&sup9;² L) — working through the exponents gives an estimate on the order of 10⁻&sup9; to 10⁻¹⁰ mol/L (roughly nanomolar to sub-nanomolar).

Conclusion: taking a human diploid cell to contain 46 DNA molecules (one per chromosome) within a cell volume of roughly half a picolitre gives an extremely low molar DNA concentration, on the order of nanomolar or less — illustrating just how few, yet how enormous, the DNA molecules inside a single cell actually are.

No — restriction endonucleases are, as far as this chapter describes, a bacterial (prokaryotic) phenomenon, not a natural feature of eukaryotic cells.

1. As the chapter explains, restriction endonucleases were discovered as enzymes that restrict the growth of bacteriophage in E. coli — that is, they form part of a bacterium’s own natural defence system against invading viral (phage) DNA.
2. A bacterium protects its own DNA from its own restriction enzyme by chemically modifying (methylating) its DNA at the recognition sequences; any unmodified, foreign DNA entering the cell — such as phage DNA — is recognised and cut, ‘restricting’ the phage’s ability to multiply. This restriction–modification system is precisely why the enzyme is called a restriction enzyme in the first place.
3. Eukaryotic cells are not known to use this particular restriction-modification strategy as their defence mechanism against foreign/viral DNA; they instead rely on entirely different, more complex systems (such as the adaptive immune system in animals). Restriction endonucleases of the type used in genetic engineering have been isolated specifically from bacteria (over 230 bacterial strains, as stated in this chapter).

Conclusion: restriction endonucleases are a bacterial defence mechanism against invading phage DNA, protecting the bacterium’s own (methylated) DNA while destroying unmodified foreign DNA — a system that, as described in this chapter, is characteristic of bacteria and is not how eukaryotic cells are known to defend themselves against foreign DNA.

Beyond aeration and mixing, stirred-tank bioreactors offer several further advantages over laboratory shake flasks:

1. Large-scale volume handling. Bioreactors can process 100–1000 litres of culture at a time, producing far greater biomass and product yield than the small volumes possible in a shake flask, which is restricted to lab-scale quantities.
2. Dedicated environmental control systems. A stirred-tank bioreactor is equipped with a temperature control system, a pH control system, an oxygen delivery system, and a foam control system — none of which a simple shake flask has — allowing the growth conditions to be kept at their precise optimum throughout the run.
3. Continuous monitoring without disturbing the culture. Bioreactors have sampling ports that allow small volumes of the culture to be withdrawn periodically for testing, so progress can be monitored without having to stop or disturb the whole batch, which is not practical with a shake flask.
4. Sustained high-yield growth. Bioreactors can be run as a continuous culture system, with used medium drained from one side and fresh medium added from the other, keeping cells in their physiologically most active (exponential/log) growth phase for longer, producing a larger biomass and higher product yield than the batch-limited conditions of a shake flask.

Conclusion: beyond better aeration and mixing, stirred-tank bioreactors offer large-scale volume capacity, dedicated temperature/pH/oxygen/foam control systems, non-disruptive sampling ports, and the ability to sustain cells in continuous, high-yield exponential growth — all advantages a simple shake flask cannot provide.

A DNA palindrome is a sequence of base pairs that reads the same on both strands when read in the 5′→3′ direction — exactly as NCERT illustrates with GAATTC/CTTAAG. The recognition sequences of most restriction enzymes are palindromic, giving us five ready examples:

Creating a new palindromic sequence by following base-pairing rules:

1. Choose any sequence for the first half, say GGT.
2. Find its complementary bases, base by base: G→C, G→C, T→A, giving CCA.
3. Reverse this complement: ACC.
4. Join the original half with this reversed complement: GGT + ACC = GGTACC.

Checking this worked example, GGTACC, against its complementary strand (CCATGG, read 3′→5′, i.e., GGTACC read 5′→3′ on that strand too) confirms it reads identically on both strands — it is indeed a true DNA palindrome (and, in fact, the real recognition sequence of the enzyme KpnI).

Conclusion: restriction-enzyme recognition sites such as GAATTC, GGATCC, AAGCTT, CTGCAG and GTCGAC are all well-known DNA palindromes, and any new palindromic sequence can be constructed by taking the reverse complement of a chosen first half and appending it to form the second half.

Natural genetic recombination occurs during meiosis I, specifically in the pachytene sub-stage of Prophase I.

1. During pachytene, the paired homologous chromosomes (bivalents, formed during the earlier zygotene sub-stage) are held together by a synaptonemal complex, and crossing over takes place between non-sister chromatids of the homologous chromosomes.
2. This crossing over is mediated by the enzyme complex recombinase and results in the physical exchange of genetic material (segments of DNA) between the maternal and paternal chromatids — this exchange is what constitutes natural genetic recombination.
3. The points of crossing over appear microscopically, in the next sub-stage (diplotene), as X-shaped structures called chiasmata.

Conclusion: recombinant DNA, in the natural biological sense, is produced during the pachytene sub-stage of Prophase I of meiosis, when crossing over between non-sister chromatids of homologous chromosomes exchanges genetic material, visible later as chiasmata in diplotene.

A reporter enzyme is an enzyme whose activity (or loss of activity) can be directly observed — usually as a visible colour change — and can therefore ‘report’ whether a piece of foreign DNA has been successfully inserted, without needing to grow cells on two different antibiotic plates.

1. In this chapter, the gene for the enzyme β-galactosidase is used as a cloning site within the vector. Recombinant DNA (the foreign gene of interest) is inserted within the coding sequence of the β-galactosidase gene.
2. If the insert is present, the β-galactosidase gene is disrupted — a phenomenon called insertional inactivation — and the enzyme is no longer functional.
3. When bacterial colonies are grown in the presence of a chromogenic substrate, colonies carrying a plasmid without an insert still have functional β-galactosidase and turn the substrate blue; colonies carrying a plasmid with the insert (recombinants) have no functional enzyme, so the colonies remain colourless (white).
4. This gives a direct, single-plate, visual (colour-based) way to distinguish recombinant from non-recombinant colonies — simpler than the two-antibiotic-plate method used for an ordinary selectable marker — and can be used alongside a normal antibiotic-resistance selectable marker (which first confirms the cell took up the plasmid at all) to doubly confirm that the plasmid taken up is specifically the recombinant one.

Conclusion: a reporter enzyme like β-galactosidase monitors transformation by changing colour only when it remains intact — insertion of foreign DNA within its gene causes insertional inactivation, turning recombinant colonies colourless against the blue non-recombinant colonies, giving a simple visual read-out that complements the antibiotic-resistance selectable marker.

Conclusion: the origin of replication lets a DNA fragment multiply inside a host, the bioreactor is the vessel in which that host is grown at scale to manufacture the desired product, and downstream processing is everything done afterwards to purify, formulate and quality-test that product before it reaches the market.

(a) PCR (Polymerase Chain Reaction). PCR is a technique for synthesising multiple copies of a gene (or any DNA segment) of interest in vitro, using two sets of primers (short, chemically synthesised oligonucleotides complementary to the flanking regions of the target DNA) and the enzyme DNA polymerase. Each cycle has three steps — denaturation (heating to separate the double-stranded DNA into single strands), primer annealing (primers bind to their complementary sequences on the single strands) and extension (DNA polymerase extends the primers using free nucleotides, copying the template). Because a thermostable DNA polymerase from the bacterium Thermus aquaticus is used, it survives the repeated high-temperature denaturation steps, and repeating the cycle many times can amplify the DNA segment to roughly a billion copies.

(b) Restriction enzymes and DNA. Restriction enzymes (restriction endonucleases) are ‘molecular scissors’ that recognise a specific, short palindromic sequence of base pairs in DNA (its recognition sequence) and cut both strands of the double helix at a fixed point within or near that sequence, in each strand’s sugar-phosphate backbone. Because the cut is offset from the centre of symmetry, it leaves short, complementary single-stranded overhangs known as sticky ends, which can hydrogen-bond with any other DNA fragment cut by the same enzyme — the basis of constructing recombinant DNA using DNA ligase.

(c) Chitinase. Chitinase is the enzyme used to break open the chitin-containing cell wall of fungal cells during the isolation of genetic material (DNA). Since DNA is enclosed within cell membranes/walls, the appropriate cell-wall-digesting enzyme must be used depending on the source organism — lysozyme for bacteria, cellulase for plant cells, and chitinase for fungi — to release the DNA along with other macromolecules before it is purified.

Conclusion: PCR amplifies a chosen DNA segment exponentially through repeated denaturation–annealing–extension cycles, restriction enzymes cut DNA at specific palindromic sites to generate sticky-ended fragments for recombination, and chitinase is the specific enzyme used to break open fungal cell walls when isolating DNA from fungal tissue.

(a) Plasmid DNA vs Chromosomal DNA

(b) RNA vs DNA

(c) Exonuclease vs Endonuclease

Conclusion: plasmid DNA is distinguished from chromosomal DNA by its small, autonomously-replicating, extra-chromosomal nature; RNA is distinguished from DNA both structurally (ribose sugar, uracil, single strand) and experimentally (selective digestion by ribonuclease); and exonucleases are distinguished from endonucleases by acting at the ends of DNA versus at specific internal positions.