Biotechnology

Class 12 Biology, unit 10, book pages 189–209

Practise 63 MCQs Take the unit quiz Review 54 cards

Biotechnology is the use of living organisms, or their processes and products, for human welfare. The unit concentrates on genetic engineering: how recombinant DNA is made with restriction enzymes, plasmid vectors and DNA ligase, how human insulin is produced in bacteria, how the polymerase chain reaction copies DNA in a machine, and what genetically modified organisms are and why people worry about them. It ends with soil-free vertical food farms and a comparison with the farming practised in Pakistan.

Learning outcomes (8)

Introduce genetic engineering.
Explain polymerase chain reaction (PCR).
Outline the function of restriction enzymes.
Describe plasmid as vector prokaryotes and explain how recombinant plasmids can be formed.
Define genetically modified organism.
Explain the formation of human insulin protein in bacteria.
Describe how vertical food farms (soil free) work.
Compare and contrast the advantages of vertical food farms with general agricultural practices prevalent in Pakistan.

Biotechnology

The history of biotechnology is as old as human civilisation. Early people realised that they could plant their own crops and breed and domesticate their own animals, so that good food was always available. The word biotechnology itself was introduced only at the end of the 20th century.

Biotechnology: The use of living organisms, especially microorganisms, or their processes and products for the welfare of mankind.

Biotechnology is therefore not new. Humans have used it for a very long time, long before anyone knew about DNA or genes. The book gives these old examples:

Yogurt and cheese are made from fermented milk.
Wine and beer are made from fermented juices of fruit, sugarcane or malted barley.
Leather has been prepared since early civilisations by the tanning process, which involves the use of multiple bacteria.

Traditional uses of biotechnology named in the book.
Product	Raw material	What is used
Yogurt and cheese	Milk	Fermentation (by microorganisms)
Wine or beer	Juice of fruit, sugarcane or malted barley	Fermentation (by microorganisms)
Leather	Animal skin	Tanning, using multiple bacteria

10.1Introduction to genetic engineeringOften examined

The needs of a rapidly growing human population led humans to develop new animals with modified characteristics. Biologists also imagined growing more nutritious and naturally pest-resistant crops. Biotechnology is a very large field, so this chapter touches only a brief part of it, called genetic engineering.

Genetic engineering: The manipulation or alteration of the genetic material of an organism to modify its characteristics or capabilities for human welfare. It is done by adding, removing or editing specific genes within the genome of the organism.
Recombinant DNA technology: The technique on which genetic engineering is based. It involves combining DNA (genes) from different sources to create new genetic combinations.

The goal is to give the organism new traits, to improve its existing traits or to eliminate undesirable traits. Humans hope to develop unlimited and useful medicines and vaccines by modifying the genomes of microorganisms. Bacteria have already been used to clean up environmental pollutants, to increase the fertility of the soil and to kill insect pests.

Major fields of application

Applications of genetic engineering.
Field	Application given in the book
Agriculture	Genetically modified (GM) crops are produced that are resistant to pests, diseases or adverse environmental stresses.
Medicine	Research on gene therapies for genetic disorders; innovative and effective ways to combat fatal diseases such as hepatitis and cancer; improved biopharmaceuticals such as insulin, antibiotics and vaccines are synthesised by genetic engineering.
Industry	Genetic engineering of microorganisms helps in the production of biofuels, the biodegradation of industrial and sewage waste, and the development of environment friendly chemicals.

The three major fields in which genetic engineering is applied, with the book's example for each.

Genetic engineering is now one of the fastest growing areas of science. The great genetic engineer Craig Venter rightly said that the 21st century is the "Century of Biology". Biotechnology has been included among the six priority areas of science and technology by the National Commission of Science and Technology of Pakistan.

10.1.1Recombinant DNA technologyOften examined

Recombinant DNA technology is the foundation of genetic engineering. It is a series of steps that join DNA segments from different sources. It is an in vivo (in living cells) method, used for the synthesis of the product of a gene, besides making copies of the desired gene (the gene of interest), on an industrial scale.

The technology involves selecting and isolating the gene of interest, inserting it into a suitable vector, and transforming a suitable host with the recombinant DNA (rDNA).

Recombinant DNA technology: A series of steps that join DNA segments from different sources. It involves the selection and isolation of the gene of interest, its insertion into a suitable vector and the transformation of a suitable host by the recombinant DNA (rDNA).
In vivo: In living cells.
In vitro: Latin for "in glassware": outside the living cell.

The components or requirements of recombinant DNA technology are:

The five components of recombinant DNA technology.
Component	What it is	Section
i. Gene of interest	The gene that is to be cloned	this section
ii. Molecular scissors	Restriction endonucleases, which cut DNA	10.2
iii. Molecular carrier or vector	A vehicle (for example a plasmid) that carries the gene into the host	10.3
iv. Molecular glue	DNA ligase, which joins the pieces	10.3.2
v. Expression system	The host cell or organism in which the recombinant DNA is cloned or expressed	10.3.2

Overview of recombinant DNA technology. The five components (gene of interest, molecular scissors, vector, molecular glue and expression system) are used across these four steps.

Gene of interest

The gene of interest is the gene which is to be cloned. It can be obtained in one of three possible ways:

Obtaining the gene of interest

Artificial gene synthesis. The gene is synthesised in vitro (outside the living cell) with the help of a DNA synthesizer machine, without any template DNA sample.
From mRNA. The gene is made from its mRNA by the enzyme reverse transcriptase, which is naturally found in retroviruses. The DNA formed by this process is called complementary DNA (cDNA).
Direct cleavage. In most cases the gene of interest is cleaved directly from a chromosomal DNA by using particular DNA scissors called restriction endonucleases.

Three ways of obtaining the gene of interest. In each case the product is a piece of DNA that carries the gene to be cloned.

10.2Molecular scissors (restriction endonuclease)Often examined

Restriction endonucleases are enzymes that cleave the phosphodiester bonds of both strands of a DNA duplex at specific sequences. They are used to cut the gene of interest during genetic recombination. In recombinant DNA technology they are called the molecular scissors.

Restriction endonuclease: An enzyme that cuts both strands of DNA at a specific sequence, the recognition (restriction) site, by breaking phosphodiester bonds.
Recognition site: The specific sequence, also called a restriction site, at which a restriction endonuclease cuts.
Palindromic sequence: A sequence of four to eight base pairs in DNA in which the nucleotides are arranged symmetrically in reverse order in both complementary strands.
Sticky ends: Single-stranded projecting ends left on the duplex fragments when a restriction enzyme makes a staggered cut.
Methylation: The addition of a methyl (CH₃) group to the nucleotides of a recognition sequence. It is carried out by the modifying enzyme methyltransferase.

Why bacteria have restriction endonucleases

Restriction endonucleases are the host-defence system of a bacterial cell against infecting viruses. The name is given because the enzyme is naturally found inside the bacterial cell to "restrict" the growth of infecting viruses by digesting (chopping up) the DNA or RNA of the viruses at specific sequences called restriction sites or recognition sites.

How a bacterium uses restriction endonucleases

A virus (bacteriophage) infects the bacterium and injects its DNA into the cell.
The restriction endonuclease recognises its restriction sites in the viral DNA and cuts both strands there. The viral DNA is chopped into pieces, so the infection is restricted.
The bacterium protects its own DNA by modifying its own restriction sites, generally by adding a methyl (CH₃) group to the nucleotides in the recognition sequences.
Methyl groups block the enzyme. They stop the endonuclease from cutting the host DNA, so the bacterial DNA is protected from its own restriction enzymes. Viral DNA is usually unmethylated, so only the viral sites are cleaved.

Figure 10.1: Functioning of restriction endonucleases in a bacterial cell. The enzyme (E) cuts the injected viral DNA at its restriction sites; the methylated host DNA is not cut.

History and naming

In 1970 the first restriction enzyme was isolated. Many different restriction endonucleases have been isolated since. Generally an endonuclease is named after the genus, species and strain of the bacterium from which it was first isolated.

Examples of restriction enzymes given in the book and the bacteria they were isolated from.
Enzyme	Bacterium it came from	Genus, species, strain
EcoRI	Escherichia coli	Escherichia, coli, strain R
Hind-II and Hind-III	Haemophilus influenzae	Haemophilus, influenzae, strain d
XhoI	Xanthomonas holcicola	Xanthomonas, holcicola

Palindromic sequences and the two kinds of cut

Restriction sites are recognised by the endonuclease because of specific palindromic sequences. A palindromic sequence is four to eight base pairs long, and the nucleotides are arranged symmetrically in reverse order in both complementary strands. For example, the sequence GAATTC reads 5′ to 3′ as GAATTC on one strand and also as GAATTC on the complementary strand.

Restriction enzymes make either a staggered cut or a blunt cut. A staggered cut gives duplex fragments that show single-stranded projecting ends called sticky ends. A blunt cut gives fragments with no sticky ends.

Staggered cut and blunt cut.
Point	Staggered cut	Blunt cut
Cut	The two strands are cut at different positions	Both strands are cut at the same position, straight across
Ends of the fragments	Single-stranded projecting (sticky) ends	Flat ends with no projection
Examples	EcoRI (sticky ends at the 5′ ends of both strands); KpnI (sticky ends at the 3′ ends of both strands)	HaeIII

Figure 10.2: EcoRI recognises the palindromic sequence GAATTC and cuts it in a staggered way, producing sticky ends at the 5′ end of both strands. The single-stranded overhangs are shown in accent.

Figure 10.3: HaeIII recognises the palindromic sequence GGCC and cuts straight through both strands, producing blunt ends with no sticky overhang.

Worked example: is a sequence palindromic?

A restriction enzyme recognises the sequence 5′-CTGCAG-3′. Show that it is a palindromic sequence. Then test 5′-GATTAC-3′.

Write the complementary strand under the given strand, base by base (A pairs with T, G with C). This strand runs 3′ to 5′. For CTGCAG it is GACGTC.
Read the complementary strand in the 5′ to 3′ direction, that is, backwards: CTGCAG.
Compare. CTGCAG is the same as the original, so the sequence reads the same 5′ to 3′ on both strands. It is palindromic (6 base pairs, within the four to eight of the book).
Repeat for GATTAC. Complement (3′ to 5′): CTAATG. Read 5′ to 3′: GTAATC.
GTAATC is not the same as GATTAC, so GATTAC is not palindromic and an enzyme that needs a palindromic site will not cut it.

Sequence 5′ to 3′	Complement 3′ to 5′	Complement read 5′ to 3′	Palindromic?
CTGCAG	GACGTC	CTGCAG	Yes
GATTAC	CTAATG	GTAATC	No

C T GC A G i s p a l in d r o mi c; G A T T A C i s n o t .

Worked example: counting the fragments

A restriction enzyme has three recognition sites in a linear DNA molecule and three recognition sites in a circular plasmid. How many fragments does it give from each?

In a linear DNA each cut separates the molecule into one more piece, so the first cut gives 2 fragments, the second 3 and the third 4. Number of fragments = number of cuts + 1.
A circular DNA has no ends. The first cut only opens the circle into one linear piece, the second cut makes 2 pieces and the third makes 3. Number of fragments = number of cuts.
Check with the figure: the linear molecule gives A, B, C and D, and the plasmid gives P, Q and R.

Cut sites (arrows) on a linear DNA and on a circular plasmid, and the fragments formed.

L in e a r D N A : 4 f r a g m e n t s . C i r c u l a r pl a s mi d : 3 f r a g m e n t s .

10.3Molecular carriers or vectorsOften examined

For the synthesis of recombinant DNA (rDNA), a molecular vector is needed to transfer the recombinant DNA into a host cell. A vector acts as a vehicle that carries foreign DNA (the gene of interest) into a host cell for multiplication and for its expression, that is, for the production of a specific protein such as insulin.

Vector (molecular carrier): A vehicle that carries foreign DNA (the gene of interest) into a host cell for its multiplication and expression.
Plasmid: A small circular DNA molecule naturally found in bacterial cells. It is separate from the chromosomal DNA and can replicate independently.

One common type of vector is a plasmid. Other vectors named in the book are listed below.

Vectors other than plasmids.
Vector	What it is
Lambda phage DNA	DNA of the bacteriophage lambda
Cosmid	A combination of plasmid and phage DNA
Yeast artificial chromosomes (YACs)	Artificial chromosomes used in yeast
Bacterial artificial chromosomes (BACs)	Artificial chromosomes used in bacteria

Plasmids

Plasmids were discovered by investigators studying the sex life of the intestinal bacterium Escherichia coli.
They are primarily found in bacterial cells but can also be found in some eukaryotes, for example yeast and some higher plants.
They are separate from the chromosomal DNA, so they are called extrachromosomal, and they can replicate independently.
They carry genes for antibiotic resistance and fertility, among others.
One of the common plasmids discovered earlier, pSC-101, has an antibiotic resistance gene for tetracycline, whereas pBR-322 has antibiotic resistance genes for both tetracycline and ampicillin.

Characteristics of a good plasmid vector

Plasmids are commonly used as vectors in genetic engineering because they have the following characteristics, which are essential for a vector:

Six characteristics of a plasmid vector.
Characteristic	What it does
i. Origin of replication site	The plasmid contains an origin of replication (Ori) site, which is required for self-regulated replication of the plasmid in the host cell. Plasmids are therefore ideal vectors for producing multiple copies of an inserted gene.
ii. Antibiotic resistance genes as selectable marker	Markers help to select the cells (bacteria) that have successfully taken up the recombinant plasmid. Plasmids usually contain antibiotic resistance genes (for example for ampicillin or tetracycline). A bacterium that has these genes is resistant to that specific antibiotic.
iii. Restriction sites of different enzymes	A vector should have a restriction site for the commonly used endonucleases; at least one restriction site is required for inserting the foreign DNA. Unique restriction sites give flexibility in the choice of endonuclease for easy insertion of foreign DNA.
iv. Promoter regions for gene expression	Plasmids may contain promoter sequences that enable transcription of the inserted genes in the host cell, so the gene of interest is expressed.
v. Small size	Plasmids are much smaller than chromosomal DNA, so they are easily purified as intact molecular vectors and easily inserted into host cells.
vi. High copy number	Some plasmids are found in high copy numbers inside the host cell, giving large amounts of the cloned DNA.

Main features of a plasmid used as a vector. The antibiotic resistance gene (accent) is the selectable marker.

Types of plasmids as vectors

Types of plasmids as vectors (Science tidbits).
Type	Use	Contains
Cloning vectors	Used for cloning of DNA fragments	Essential elements such as Ori, selectable markers and cloning sites
Expression vectors	Designed for the expression of a gene in a host organism	Promoters, ribosome binding sites and termination sequences
Shuttle vectors	Plasmids that can replicate in multiple host species, for example in both prokaryotic and eukaryotic cells, so they are useful for gene transfer between different biological systems	(replicate in more than one host)

Applications of plasmids

Applications of plasmids (Science tidbits).
Application	What plasmids do
Gene cloning	Extensively used for cloning of genes of interest for further study and manipulation.
Protein production	Expression vectors are used for the production of proteins by expressing the inserted genes.
Gene therapy	Used to deliver therapeutic genes into cells of organisms with a genetic disease.
Vaccine development	Most DNA vaccines are plasmid based and are effectively used to prevent diseases.

10.3.1pBR322 as an example of plasmids

pBR322 is the first and most common purpose-built plasmid vector, artificially developed in the laboratory by Bolivar and Rodriguez in 1977. It is modified from a natural bacterial plasmid. This vector has an origin of replication (ori), restriction sites and also antibiotic-resistance genes.

Key features of the pBR322 plasmid

Key features of pBR322. Features i to iv are from the book's list; the resistance genes are shown in its Figure 10.4 and described in section 10.3.
Feature	Description
i. Origin of replication	It carries a fragment of the plasmid pMB1 that acts as an origin for DNA replication, to ensure the multiplication of the vector.
ii. Size	It is a relatively small plasmid of 4,361 base pairs. Small size is important because transformation efficiency is inversely proportional to the size of the plasmid. Transformation efficiency becomes very low if the plasmid size is above 10 kbp, so a DNA segment of up to 6 kbp can possibly be inserted in pBR322 to form the recombinant.
iii. Copy number	It has a relatively high copy number (about 15 copies per cell).
iv. Cloning sites	It carries a number of unique restriction sites. Some are located in one of the antibiotic resistance genes: sites for PstI, PvuI and SacI are found in the ampicillin gene, and BamHI and HindIII in the tetracycline gene.
v. Antibiotic resistance	It has genes for resistance to both ampicillin and tetracycline.

Figure 10.4: The E. coli cloning vector pBR322 plasmid has an origin of replication (ori), restriction sites and antibiotic-resistance genes (accent).

10.3.2Mechanism of formation of recombinant plasmid in recombinant DNA technologyOften examined

Cloning of the desired gene by recombinant DNA technology involves three things: the formation of the recombinant DNA (gene of interest + vector DNA), transformation into a suitable expression system by the recombinant DNA, and identification of the transformed clones.

Formation of recombinant plasmid

Making the recombinant plasmid

Isolate and purify the gene of interest and the relevant vector. The gene is cleaved directly from the genome by specific restriction endonucleases (leaving sticky ends), or it may be synthesised from mRNA by reverse transcriptase.
Cut the vector. The plasmid, which has the same restriction site, is cleaved with the same restriction enzyme, so that compatible sticky ends are produced on the gene and the vector.
Mix and anneal. The vector and the gene of interest are incubated together; their complementary sticky ends pair up (anneal).
Join with DNA ligase. DNA ligase connects them by forming phosphodiester linkages. When the gene of interest is ligated to the plasmid at the complementary sticky ends, the resulting plasmid is called a recombinant plasmid (vector).

Figure 10.5: Steps for the formation of recombinant plasmid. The gene of interest (accent) is cut from human DNA, ligated into the cut plasmid, and the recombinant plasmid is taken up by a host cell.

Making the recombinant plasmid, step by step. The same restriction enzyme cuts the human DNA and the plasmid, the sticky ends of the gene of interest (accent) pair with those of the cut plasmid, DNA ligase seals the joints, and the recombinant plasmid is taken up by the bacterium.

Molecular glue (DNA ligase): The enzyme responsible for forming the phosphodiester linkage between two adjacent nucleotides. It joins two double-stranded DNA fragments, so it is called molecular glue. In rDNA work it joins the plasmid (vector) and the foreign DNA that are annealed by their sticky ends.
Expression system: A suitable cell or organism that can act as host for the recombinant vector, for its cloning (multiplication) or expression.
Transformation: The insertion of recombinant DNA into the expression system.
Transformed clone: A bacterial clone that has taken up the recombinant plasmid; every cell of the clone contains at least one plasmid carrying the gene of interest.

Products of recombinant DNA technology can be produced by using a suitable expression system. Plasmids are usually made recombinant for the production of a specific protein as a result of expression of the gene of interest in a natural expression system.

Expression system

The choice of expression system always depends upon the type of vector being used. The most important character of an ideal expression system is its short generation time and simplicity of its cellular and genetic system. For this reason bacterial cells act as an ideal expression system.

Transformation of the expression system

Transformation

Mix. Bacterial cells that already contain no plasmids (the expression system) and the recombinant plasmids are put into the same medium.
Uptake. The bacterial cells take up the recombinant plasmid, especially if they are treated with calcium chloride, which makes them more permeable.
Cloning. As the cell reproduces, a bacterial clone forms and each new cell contains at least one plasmid. So each bacterial cell contains the gene of interest, which will express itself and make its product.

Identification of the transformed clone

The transformed clone is identified by adding a particular antibiotic (the one for which a resistance gene is present in the plasmid) to the medium. The transformed clone has resistance against the antibiotic, so it remains alive and continues to grow, whereas all the untransformed clones are killed by the antibiotic. From this transformed clone the cloned gene can be isolated for further analysis, or its protein product can be separated and used for various purposes.

Worked example: who survives on the antibiotic plate?

A student treats E. coli cells with calcium chloride and mixes them with a recombinant plasmid that carries the gene of interest and an ampicillin resistance gene. The mixture is spread on a medium containing ampicillin. Predict what happens to (a) cells that took up the plasmid and (b) cells that did not, and explain why this identifies the transformed clone.

Ampicillin is added to the medium because the plasmid carries a resistance gene for it. The gene is the selectable marker.
(a) A cell that took up the recombinant plasmid has the ampicillin resistance gene, so it survives, grows and divides. Its descendants form a clone in which every cell carries the plasmid and the gene of interest.
(b) A cell that did not take up the plasmid has no resistance gene, so the antibiotic kills it.
Only the transformed cells are alive at the end, so the colonies that grow are the transformed clone. The cloned gene or its protein can then be isolated.

Cell type	Has plasmid with resistance gene?	On ampicillin medium
Transformed	Yes	Survives and forms a clone
Untransformed	No	Killed

Transformed cells survive on the ampicillin medium; untransformed cells are killed.

10.4Formation of human insulin protein in bacteriaOften examined

Human insulin is produced by recombinant DNA technology in the following steps.

1. Identification and isolation of the insulin gene

The insulin gene, which produces human insulin, is isolated from a human DNA sample. Insulin consists of two peptide chains (α and β) linked by disulfide bonds. It is produced by the endocrine β-cells in the islets of Langerhans of the pancreas. The gene for insulin is called INS (the word insulin is derived from the Latin word insula, meaning "island").

The human insulin gene.
Fact about the insulin gene	Value
Name	INS
Location	Human chromosome 11, position 11p15.5 (short arm p, region 11, band 15, sub-band 5)
Length	1.5 kbp, about 1,425 bp
Structure	Three exons and two introns

2. Selection of a suitable plasmid for insertion of the insulin gene

The plasmids must be specially engineered to transfer genes efficiently into host cells such as Escherichia coli. Two commonly used plasmids are:

pBR322: one of the most widely used plasmids in genetic engineering. It contains antibiotic resistance genes for ampicillin and tetracycline.
pUC18/pUC19: derivatives of pBR322 that have been modified for efficient transformation. They include a gene for ampicillin resistance.

Both plasmids are small, have an origin of replication, a higher copy number and multiple unique restriction sites, which simplifies insertion of the insulin gene. Human genes for the α and β chains of insulin may be inserted into these plasmids, for expression in bacterial cells to synthesise the insulin chains.

3. Creation of recombinant DNA

Bacteria such as E. coli are not advanced cells, so they cannot directly produce eukaryotic proteins like insulin. Therefore two separate synthetic genes are created, one for the α chain and one for the β chain of insulin. The genes are inserted separately into two separate bacterial plasmid vectors (pBR322). Each gene is inserted next to the β-galactosidase gene, a highly expressed gene. The plasmids are engineered with a promoter sequence that helps to start the auto-production of the insulin chains inside the bacterial cells.

4. Insertion of recombinant plasmids into host cells

The recombinant plasmids are inserted separately into E. coli (or another bacterial strain) by transformation. The bacteria take up the recombinant plasmids and become recombinant bacteria. Transformed cells are selected by their specific antibiotic resistance.

5. Production of insulin chains

The bacteria are grown in large fermentation tanks containing a suitable culture medium under optimum conditions. As the bacteria grow, the recombinant plasmid DNA is replicated, so the insulin gene is multiplied (cloned). The bacteria also express the insulin genes and produce pro-insulin chains, that is, a fused β-galactosidase-A chain and a fused β-galactosidase-B chain, separately. Both chains are produced as part of fusion proteins, which help to protect the insulin chains from bacterial protease enzymes during synthesis.

6. Extraction and purification

The bacterial cells are harvested and the pro-insulin chains combined with fusion proteins are isolated. The pro-insulin chains A and B are separated from the fusion protein β-galactosidase by chemical treatment with cyanogen bromide. The removal is possible because an extra codon is added before the methionine codon at the N-terminal of each gene for the A and B chains.

7. Folding and disulfide bridging of both insulin chains

After detachment from the fusion proteins, the A and B chains are chemically joined in vitro to form functional insulin. Disulfide bridges at cysteine residues of both chains are formed by treating with sodium disulphonate and sodium sulphite, giving biologically active insulin.

8. Purification and testing

The insulin is further purified to remove impurities or by-products of bacterial gene expression. It is then tested for quality, effectiveness, safety and possible side effects.

9. Packaging for storage, shipment and usage

After confirmation of the best quality, insulin is packaged into vials or insulin pens, with instructions for storage and usage, for injection. Recombinant human insulin is pure, safer and much more effective than insulin previously derived from animals.

Nine steps in brief

Isolate the human insulin gene (or make two synthetic genes, for chains A and B).
Put each gene next to the β-galactosidase gene in a pBR322-type plasmid.
Transform E. coli and select the transformed cells with the antibiotic.
Grow the bacteria in fermenters; they make fusion proteins (β-galactosidase + chain).
Harvest, purify the fusion proteins and cut the chains free with cyanogen bromide.
Join chains A and B in vitro with disulfide bridges.
Purify, test, package.

From pre-pro-insulin to active insulin (Science tidbits).
Stage	Amino acids	What happens
Pre-pro-insulin	109	First polypeptide made by the β-cells; 23 amino acids act as signal molecules needed for entry through the cell membrane
Pro-insulin (inactive)	86	Inside the cell, after the 23 signal amino acids are removed (109 − 23 = 86)
Active insulin	51	Proteolytic enzymes convert pro-insulin to active insulin: chain α has 21 and chain β has 30 amino acids (21 + 30 = 51), linked by disulfide bonds

Insulin is made as pre-pro-insulin and shortened in two steps. The 23 signal amino acids (accent) are removed first, then enzymes form the two chains of active insulin. Bar length is proportional to the number of amino acids.

10.5Polymerase chain reaction (PCR)Often examined

The technique used to amplify (clone) a single gene or a piece of DNA into thousands to millions of copies by in vitro replication is called polymerase chain reaction (PCR). In this technique DNA polymerase is made to polymerise (the polymerase reaction) a given piece of DNA again and again, so that multiple copies are produced. That is why it is known as polymerase chain reaction.

Polymerase chain reaction (PCR): A technique used to amplify a single gene or a piece of DNA into thousands to millions of copies by in vitro replication.
Taq polymerase: A special, temperature-tolerant DNA polymerase isolated from Thermus aquaticus, a bacterium found in hot springs. It is stable and active at near-boiling temperatures.
Primers: Short pieces that anneal (hybridise) to the single-stranded template at its complementary regions. The forward primer and the reverse primer mark where copying begins.
Thermocycler (PCR machine): The instrument that holds the PCR mixture and regulates the temperature during the various steps of the reaction.

Components of the PCR mixture

The PCR mixture is placed in a thermocycler, which regulates the temperature during the various steps (Figure 10.7).
Component	Role
Template DNA	The DNA to be amplified
Free nucleotides (dNTPs, deoxyribonucleoside triphosphates)	Building blocks for the new strands
Primers	Start the copying at the target sequence
Taq polymerase	Heat-stable enzyme that synthesises the new strands
Suitable buffer	The medium that makes up the PCR reaction mixture

Mechanism of the PCR reaction

A PCR cycle consists of three steps: denaturation, primer annealing and extension (polymerisation). Each step needs a specific temperature. The time, temperature and sequence of the steps are programmed in the thermocycler.

Figure 10.8: Mechanism of PCR reaction. P1 is the forward primer and P2 is the reverse primer. New DNA strands (accent) are made from the primers; one cycle turns one ds DNA into two.

The steps of PCR.
Step	Temperature	Time	What happens
Initial denaturation (once, before the cycles)	94 to 95 °C	4 to 5 minutes	Completely denatures the DNA template and makes sure all strands are separated before cycling begins. GC-rich regions or complex secondary structures need a longer initial denaturation to break all the hydrogen bonds so that primers can bind properly.
Cyclic denaturation	94 °C	1 minute	Double-stranded DNA becomes single-stranded DNA; each single strand can act as a template for in vitro DNA synthesis.
Primer annealing	55 to 65 °C (depends on the size of the primer and the length of the gene)	About 2 minutes	The forward and reverse primers anneal (hybridise) to the single-stranded template at its complementary regions.
Cyclic extension (polymerisation)	72 °C	About 1 minute (1 to 2 minutes in Figure 10.9)	Taq polymerase synthesises new DNA strands at the 3′ ends of the primers using dNTPs.
Final extension (once, after the cycles)	72 °C	5 to 7 minutes	Ensures that any incomplete DNA strands left from the cycles are fully extended, giving complete double-stranded products.
Storage	4 °C	Long time	PCR products (replicated DNA copies) are stored safely until they are moved to a refrigerator.

Temperature programme of a thermocycler (schematic, not to scale)

4

55 - 65

72

94

Time

Temperature (^{\circ} C)

Initial denaturation

4-5 min

25-35 cycles

Denaturation 1 min

Annealing 2 min

Extension

1-2 min

Final extension

5-7 min

Hold 4^{\circ} C

Figure 10.9: Steps of the PCR mechanism as set in the thermocycler (PCR machine).

At the end of the first cycle, one target ds DNA molecule has become two daughter ds DNA molecules. The second cycle starts at once with denaturation at 94 °C, so all the newly made molecules are also separated into single strands, which again act as templates. Primer annealing and extension follow. The cycle of denaturation, annealing and extension continues, and the selected DNA sequence is amplified at an exponential rate: the number of existing DNA molecules is doubled after each cycle. The two non-cyclic steps, initial denaturation and final extension, contribute to the efficiency and accuracy of PCR, giving better yields and more reliable results.

Worked example: how many copies after n cycles?

One target DNA molecule is amplified by PCR. How many copies are present after 5 cycles and after 30 cycles? Explain why the increase is called exponential.

The number doubles in every cycle, so after $n$ cycles the number of copies is $2^{n}$ .
After 5 cycles: $2^{5} = 2 \times 2 \times 2 \times 2 \times 2 = 32$ copies.
After 30 cycles: $2^{30} \approx 1.07 \times 1 0^{9}$ , so more than a thousand million copies.
The increase is exponential because the number of copies is multiplied by the same factor (2) each cycle, instead of a fixed number being added. The graph curves upward more and more steeply.

Cycles ( $n$ )	0	1	2	3	4	5	10
Copies ( $2^{n}$ )	1	2	4	8	16	32	1024

Cycles ( $n$ )	Copies ( $2^{n}$ )
0	1
1	2
2	4
3	8
4	16
5	32
10	1024

200

400

600

800

1000

Number of cycles

Copies

32

1024

2^{n}

Number of copies of the target DNA against the number of PCR cycles (

2^{n}

2^{5} = 32 copies, 2^{30} \approx 1.07 \times 1 0^{9} copies

Worked example: how long does a PCR programme take?

A thermocycler is programmed with an initial denaturation of 5 minutes, then 30 cycles of denaturation 1 minute, annealing 2 minutes and extension 1 minute, then a final extension of 7 minutes (all values within the ranges in the book). Find the run time, ignoring the time taken to change temperature.

Time for one cycle = 1 + 2 + 1 = 4 minutes.
Time for 30 cycles = 30 × 4 = 120 minutes.
Add the two non-cyclic steps, which happen only once: 5 + 120 + 7 = 132 minutes.
Convert: 132 minutes = 2 hours 12 minutes, after which the machine holds the product at 4 °C.

Part of the programme	Time
Initial denaturation (once)	5 min
30 cycles × (1 + 2 + 1) min	120 min
Final extension (once)	7 min
Total	132 min = 2 h 12 min

132 min u t es (2 h o u r s 12 min u t es), t h e n s t or a g e a t 4° C .

Worked example: differentiate cloning in bacteria and PCR

Differentiate between gene cloning by recombinant DNA technology and gene amplification by PCR (three points).

Point	Recombinant DNA technology	PCR
Where it happens	In vivo, inside living host cells (bacteria)	In vitro, in a tube in a thermocycler
What copies the DNA	The host cell, which replicates the recombinant plasmid as it divides	Taq polymerase, using primers and dNTPs
Tools and steps needed	Restriction endonuclease, vector, DNA ligase, transformation and selection with antibiotic	Template DNA, primers, dNTPs, Taq polymerase and repeated heating and cooling cycles (denaturation, annealing, extension)

The point of comparison is the main idea: one uses living cells, the other does not.
For each point write one fact about each method, side by side, as in the table.

Three-point table as above.

Why PCR is designed this way (Science tidbits questions)

Science, technology and society connections: applications of PCR

PCR is an efficient diagnostic technique used for the detection of specific genotypes of infectious agents.
Reactions for DNA sequencing are also simplified by introducing the PCR method.
DNA fingerprinting is also made simple by PCR.
The genetic mutations responsible for certain genetic diseases and cancers can be detected using PCR tools. Early detection of a genetic disease is even possible in embryonic conditions or in sex cells (sperm and egg).

10.6Genetically modified organisms (GMOs)Often examined

Genetically modified organisms (GMOs) are organisms (for example bacteria) whose genetic material (DNA) has been altered in a way that does not occur naturally through fertilisation or natural recombination. Techniques of genetic engineering are used to insert foreign genes, remove existing genes or modify specific genes in naturally free living organisms to convert them into genetically modified organisms with desirable traits.

Genetically modified organism (GMO): An organism whose genetic material (DNA) has been altered in a way that does not occur naturally through fertilisation or natural recombination.
Transgenic organism: A GMO that carries a foreign gene inserted by genetic engineering. Many transgenic organisms such as animals, plants and bacteria have been produced.

GMOs have improved nutritional content and enhanced growth rates, and they are resistant to pests, diseases and environmental conditions. They are widely used in agriculture, animal husbandry, medicine and research.

Three kinds of transgenic organisms.
Type	What the book says
10.6.1 Transgenic bacteria	Unlike other organisms, bacteria can be easily transformed because of their simple genetics. The first example was in 1978, when a version of the human insulin gene was inserted into Escherichia coli to produce synthetic "human" insulin. Transgenic bacteria are used to produce different human proteins, to improve plant growth, to remove environmental pollutants and to extract metals from low grade ores.
10.6.2 Transgenic plants	The first field trials of genetically engineered plants were in France and the USA in 1986, when tobacco plants were engineered to be resistant to herbicides. In most cases the aim is to introduce a new trait that does not occur naturally in the species: resistance to certain pests, diseases or environmental conditions, or the production of a certain nutrient or pharmaceutical agent.
10.6.3 Transgenic animals	A transgenic animal carries a foreign gene that has been deliberately inserted into its genome. Genetic engineering is used to improve the traits of farm animals. These animals are also used to produce drugs. Transgenic animals that produce human proteins or drugs are called transpharmer animals.

The three kinds of transgenic organism (10.6.1 to 10.6.3) and the objective of each.

Transpharmer animals: Transgenic animals that produce human proteins or drugs.

10.6Concerns about GMOs and science, technology and society connections

Concerns about genetically modified organisms (GMOs)

The main areas of consideration for safety aspects in biotechnology are the following:

How to dispose of spent microbial biomass and purify the effluents from biotechnological processes?
The toxicity and allergy associated with microbial production.
How to deal with the increase in the number of antibiotic resistant pathogenic microorganisms?
How to evaluate the pathogenicity of the genetically engineered microorganisms to infect humans, plants and animals?
How to prevent contamination, infection or mutation of the processed strains?

Science, technology and society connections

Global transgenic crops

Since 1996 the cultivation of transgenic crops has expanded rapidly. In 2005 transgenic crops were planted globally on 90 million hectares, which increased to 190.4 million hectares by 2019. Major GM crops include herbicide-tolerant and insect-resistant soybean, maize, cotton and canola. The leading producers are the United States, Brazil, Argentina and Canada.

Transgenic crops in Pakistan

Transgenic crops in Pakistan (source quoted in the book: Discourse 2024, PIDE, Islamabad).
Crop	Facts given in the book
Bt cotton	Commercially adopted in 2010; now grown over 2.3 million hectares (95% of the cotton area).
Triple gene cotton	New varieties, resistant to pests and herbicides, introduced in 2023.
GM maize	Production increased sharply, from 1 million metric tons in 2013 to 10.5 million metric tons in 2023.
GM sugarcane	GM varieties of sugarcane have recently been approved for cultivation.

Genetic counselling, careers, accomplishments, solving problems and genetic screening

Genetic counselling: A service that provides information and advice about genetic conditions. It is conducted by healthcare professionals specially trained in the science of genetics (a genetic counsellor or a clinical geneticist), who discuss the risks, benefits and limitations of genetic testing and explain how the information from testing could have implications for you and your family.
Genetic screening: All the diagnostic tests used to determine whether a person or a newborn baby is at risk of genetic diseases.

Science, technology and society connections.
Topic from the book	What it says
Justify the need of genetic counselling	Genetic counselling is a service that provides information and advice about genetic conditions (see definition above).
Careers that need biotechnology and genetic engineering	Healthcare professionals, teachers of biological sciences, biomedical engineers, crime lab analysts, crime scene investigators, environmental impact analysts, forensic scientists, genetic engineers, molecular biologists and others.
Accomplishments of renowned genetic engineers in your province	The administration should arrange study tours of students to public and private sector institutions of genetic engineering, so that students can meet renowned genetic engineers and ask about their accomplishments directly.
Suggest measures to solve related problems using knowledge from this chapter	Students should evaluate themselves to know what they have learnt in this chapter and should apply this knowledge to solve related problems under the guidance of their class teacher.
Role of genetic screening	There are two types: screening of children and adults, and screening of unborn children. For children and adults it has two purposes: (1) it can confirm whether the person has a mutated gene for a certain disease or characteristic; (2) it can test adults to see whether their children will be at risk of a certain disease. Knowing that one or both parents carry a dominant allele for a genetic disease might affect the parents' decision about having children. Genetic screening is used for approval of marriage licences in some countries, like Denmark.

10.7Working of vertical (soil-free) food farmsOften examined

Vertical farming is soil-free agriculture in which plants are grown in vertically stacked layers or towers. It allows food production in urban areas or places with limited land. It is a common practice in densely populated areas and can be done in small spaces, including greenhouses, warehouses or empty spaces in offices and houses.

Vertical farming or soil-free farming typically means growing plants without the use of soil. Soil is heavy and may weigh down the vertical stacking system, so farmers use air and water to create a suitable artificial environment needed for plant growth. On the basis of technique and plant growing medium, soil-free farms are called hydroponics, aeroponics or aquaponics.

Vertical farming: Soil-free agriculture in which plants are grown in vertically stacked layers or towers, in a controlled artificial environment.
Hydroponics: Plants are grown with their roots submerged in a nutrient-rich water solution instead of soil.
Aeroponics: Plant roots are suspended in the air, instead of being submerged in water, and nutrient-rich water is sprayed directly onto them.
Aquaponics: Fish farming combined with hydroponics.

Figure 10.10: Working of vertical (soil-free) food farms. Plants are stacked in layers under LED lights; water and nutrients are pumped round a loop and the environment is controlled by sensors and software.

1. Hydroponics

Plants are grown with their roots submerged in a nutrient-rich water solution instead of soil. Water is used efficiently and made to flow through channels that provide nutrients directly to the plants, and it is then recycled to conserve resources, using up to 90% less water than traditional farming.

2. Aeroponics

In aeroponics the plant roots are suspended in the air instead of being submerged in water. Nutrient-rich water is sprayed directly onto the roots after specific intervals, for efficient absorption of moisture and nutrients. Aeroponics is more economical than hydroponics because it uses even less water, and it gives better oxygenation for the roots and higher crop yields.

Hydroponics and aeroponics compared: the plant is held above the roots in a support, with no soil in either method.

3. Aquaponics

Sometimes fish farming is combined with hydroponics. This is called aquaponics. Water from the fish tanks is circulated through the plant beds, where the plants absorb the nutrients. Fish excreta provide natural nutrients to the plants, and in return the plants filter the water for the fish. This creates a self-sustaining ecosystem and reduces the need for synthetic fertilisers.

Figure 10.12: Schematic diagram of aquaponics: vertical (soil-free) food farming combined with fish farming.

Three kinds of soil-free farms.
Method	Where the roots are	Water and nutrients	Special point given in the book
Hydroponics	Submerged in nutrient-rich water	Water flows through channels and is recycled	Up to 90% less water than traditional farming
Aeroponics	Suspended in air	Nutrient-rich water sprayed on the roots at intervals	Even less water than hydroponics, better oxygenation, higher yields
Aquaponics	In plant beds supplied with water from fish tanks	Fish excreta give nutrients; plants filter the water	Self-sustaining; reduces the need for synthetic fertilisers

Controlled environment agriculture

Vertical farming uses controlled environment agriculture (CEA) technology to maintain an optimal growing environment around the year. CEA includes:

vertically placed LED grow lights with inbuilt timers, to ensure the light spectrum needed for photosynthesis in plants;
temperature regulators, to ensure the optimal farm climate;
various sensors for continual monitoring of growth.

Thus soil-free farming provides faster growth and year-round production with limited resources. Vertical farming is also sustainable compared with traditional farming because it uses less water and land, releases less CO₂ and produces higher crop yields per square foot. Nowadays vertical farms are equipped with automated systems of sensors and AI support to monitor plant growth, adjust climatic conditions, adjust water and nutrient supply and optimise production.

10.8Comparison of vertical food farms with general agricultural practices in PakistanOften examined

Agriculture is the major contributor to Pakistan's economy. It provides employment to a large population, especially in rural areas, and acts as the major labour force in Pakistan. It provides raw materials for the textile, sugar, food and dairy industries of Pakistan. The main aspects of agriculture in Pakistan are:

Pakistan has 47% agricultural land, which is higher than the global average of 38%. About 82% of the cultivated land is irrigated through a well planned canal system and 18% is arid land.
The major crops are wheat, rice, sugarcane and potatoes as staple crops, and cotton and maize as main cash crops. Pakistan has two main crop seasons, Kharif and Rabi.
Livestock is complementary to agriculture and is a major contributor to the economy. Pakistan is the 5th largest in dairy products and the 4th largest exporter of leather items in the world.
Organic farming is also common, using recycled remainders of crops and animal manure, using no or minimum pesticides and relying on crop rotation, thus minimising the environmental impact.

Vertical food farms and the traditional agricultural practices prevalent in Pakistan have some advantages and challenges. Table 10.1 compares them.

Table 10.1: Comparison between vertical food farming and general agricultural practices.
Aspect	Vertical food farming	General agricultural practices
1. Space utilisation	Advantage: uses less horizontal space by stacking plants in vertical layers. Ideal for urban environments where land is limited or expensive; in big cities vertical farming is ideal because of insufficient land.	Disadvantage: requires large horizontal fields, making it less feasible in urban or densely populated areas. Crops like wheat, rice and sugarcane need vast fields, as grown in rural areas of Punjab and Sindh.
2. Water usage	Advantage: uses 90 to 95% less water than traditional farming. Hydroponic systems recycle water and aeroponics uses water spray, minimising wastage. Crucially relevant in regions facing severe water shortages, like Thar and Baluchistan.	Disadvantage: traditional methods consume large amounts of water, especially rice and sugarcane. Pakistan frequently faces drought and wastage of water due to floods in traditional irrigation systems.
3. Yield and productivity	Advantage: crops grow faster because of optimised nutrient delivery. Higher yields per square foot are possible with controlled environments (temperature, light, humidity, fertilisers) for stacked plants. Leafy greens, vegetables, herbs and small fruits like strawberry are grown throughout the year with higher production.	Disadvantage: mainly depends on seasonal cycles, weather conditions and unpredictable factors like floods and droughts. Annual productivity per unit of land remains inconsistent and lower, and crops can fail in adverse climate. Wheat production is limited to winter seasons and monsoons may damage cotton and rice crops.
4. Labour and automation	Advantage: may be easily managed by automatic and AI systems to monitor and control plant health, watering times and light durations, so the need for manual labour is minimal. Disadvantage: the high cost of automation may be a barrier to making it common, especially in rural areas of Pakistan.	Disadvantage: mainly depends on manual labour and mostly uses outdated tools and techniques. Less use of modern and efficient machinery leads to low productivity.
5. Crop varieties	Disadvantage: suited only for short-cycle crops like leafy green vegetables, herbs and small fruit plants like strawberries. Large scale staple crops (wheat, rice, maize) may not be easily grown in vertical systems because of land usage challenges.	Advantage: can produce a wide range of crops, including grains (wheat, rice, maize), fruits (mangoes, apples, oranges) and vegetables like potatoes. Traditional agriculture provides food security.
6. Pesticide and chemical usage	Advantage: rare chances of insects and pests in controlled environments, so minimal need for pesticides and herbicides, giving pesticide and toxin free food. It may improve food quality and safety by avoiding pesticide contamination in agricultural zones.	Disadvantage: frequent and extensive use of pesticides and fertilisers is needed to avoid loss of food to insects and pests. Improper handling of toxins may harm human health and lead to environmental degradation.
7. Environmental impact	Advantage: usually located in cities, so negative impacts on the environment are rare. It is sustainable because of less usage of water and land, minimal need for transportation, negligible fossil fuel emissions and no soil erosion. However, the energy demand of LED grow lights and climate control may be managed by using renewable energy like solar electricity.	Disadvantage: greatly at risk of being affected by environmental hazards like soil degradation, salinity, floods and drought. Another issue is deforestation, due to the desire for expanding agricultural land. Extreme climate changes have often deteriorated traditional agriculture and resulted in loss of agricultural land.
8. Accessibility and investment	Disadvantage: requires higher initial investment to establish infrastructure for lighting, climate control and automation, so it is less accessible to small farmers in Pakistan. However, it may be ideal for agriculture farming business in urban areas.	Advantage: lower initial investment in many cases, particularly for small-scale farmers who rely on simple tools and natural resources. Disadvantage: yields are generally lower, and dependence on natural weather cycles makes it unpredictable.

Quick summary of Table 10.1.
Aspect	Vertical farming	General agriculture
Space	Little land needed (stacked layers)	Large fields needed
Water	90 to 95% less water	Large amounts of water
Yield	Year-round, higher per area	Seasonal, weather-dependent
Labour	Automated, little manual labour	Manual labour, outdated tools
Crop range	Only short-cycle crops	Grains, fruits and vegetables
Pesticides and environment	Few pesticides, low impact	More pesticides, climate and soil risks
Cost	High initial investment	Low investment for small farmers

Key terms

Biotechnology: The use of living organisms, especially microorganisms, or their processes and products for the welfare of mankind.
Genetic engineering: The manipulation or alteration of the genetic material of an organism to modify its characteristics or capabilities for human welfare, by adding, removing or editing specific genes.
Recombinant DNA technology: A series of steps that join DNA segments from different sources: selection and isolation of the gene of interest, insertion into a suitable vector and transformation of a suitable host by the recombinant DNA (rDNA).
In vivo: In living cells.
In vitro: Latin for "in glassware": outside the living cell.
Gene of interest: The gene that is to be cloned.
Artificial gene synthesis: Synthesis of a gene in vitro, without template DNA, with the help of a DNA synthesizer machine.
Reverse transcriptase: The enzyme, naturally found in retroviruses, that makes DNA from mRNA.
Complementary DNA (cDNA): The DNA formed from mRNA by reverse transcriptase.
Restriction endonuclease: An enzyme that cleaves the phosphodiester bonds of both strands of DNA at specific sequences; the molecular scissors and a host-defence system of bacteria against viruses.
Recognition site (restriction site): The specific sequence at which a restriction endonuclease cuts.
Palindromic sequence: A sequence of four to eight base pairs in DNA in which the nucleotides are arranged symmetrically in reverse order in both complementary strands.
Staggered cut: A cut in which the resulting duplex fragments show single-stranded projecting ends called sticky ends.
Sticky ends: Single-stranded projecting ends of duplex fragments formed by a staggered cut.
Blunt cut: A cut in which the resulting duplex fragments do not show sticky ends.
Methylation: Addition of methyl (CH₃) groups to the nucleotides in the recognition sequences of the bacterium's own DNA, carried out by the enzyme methyltransferase.
Vector (molecular carrier): A vehicle that carries foreign DNA (the gene of interest) into a host cell for multiplication and expression.
Plasmid: A small circular DNA molecule naturally found in bacterial cells, separate from the chromosomal DNA and able to replicate independently.
Cosmid: A vector that is a combination of plasmid and phage DNA.
Yeast artificial chromosomes (YACs): Artificial chromosomes used as vectors in yeast.
Bacterial artificial chromosomes (BACs): Artificial chromosomes used as vectors in bacteria.
Origin of replication (Ori): The site on a plasmid required for self-regulated replication of the plasmid in the host cell.
Selectable marker: A gene, usually for antibiotic resistance, that helps in selecting the cells that have taken up the recombinant plasmid.
pBR322: The first and most common purpose-built plasmid vector (4,361 bp), developed by Bolivar and Rodriguez in 1977, with an origin of replication, restriction sites and genes for resistance to ampicillin and tetracycline.
Cloning vector: A plasmid used for cloning DNA fragments; contains Ori, selectable markers and cloning sites.
Expression vector: A plasmid designed for the expression of a gene in a host organism; includes promoters, ribosome binding sites and termination sequences.
Shuttle vector: A plasmid that can replicate in multiple host species, for example in both prokaryotic and eukaryotic cells.
DNA ligase (molecular glue): The enzyme that forms the phosphodiester linkage between two adjacent nucleotides and joins two double-stranded DNA fragments.
Recombinant plasmid: A plasmid in which the gene of interest has been ligated at the complementary sticky ends.
Expression system: A suitable cell or organism that acts as host for the recombinant vector for its cloning or expression.
Transformation: The insertion of recombinant DNA into the expression system.
Transformed clone: A clone of bacterial cells that took up the recombinant plasmid and can be identified by the antibiotic for which the plasmid carries resistance.
INS: The human insulin gene, located on chromosome 11 at 11p15.5.
Fusion protein: A protein in which an insulin chain is joined to β-galactosidase; it protects the chain from bacterial proteases.
Cyanogen bromide: The chemical used to separate the insulin chains from the β-galactosidase fusion protein.
Polymerase chain reaction (PCR): A technique to amplify a single gene or piece of DNA into thousands to millions of copies by in vitro replication.
Taq polymerase: A temperature-tolerant DNA polymerase isolated from Thermus aquaticus, stable and active at near-boiling temperatures.
Primers: Forward and reverse primers that anneal to the single-stranded template at its complementary regions to start DNA synthesis in PCR.
dNTPs: Deoxyribonucleoside triphosphates, the free nucleotides used in PCR.
Thermocycler (PCR machine): The instrument that regulates the temperature during the steps of PCR.
Denaturation: The PCR step at 94 °C in which double-stranded DNA becomes single-stranded.
Primer annealing: The PCR step at 55 to 65 °C in which the primers hybridise to the template.
Extension (polymerisation): The PCR step at 72 °C in which Taq polymerase synthesises new DNA strands from the 3′ ends of the primers.
Genetically modified organism (GMO): An organism whose genetic material has been altered in a way that does not occur naturally through fertilisation or natural recombination.
Transgenic organism: An organism carrying a foreign gene deliberately inserted into its genome, such as a transgenic bacterium, plant or animal.
Transpharmer animals: Transgenic animals that produce human proteins or drugs.
Bt genes: Pest-resistance genes from the bacterium Bacillus thuringiensis, used in transgenic crops.
Genetic counselling: A service that provides information and advice about genetic conditions.
Genetic screening: Diagnostic tests used to determine whether a person or a newborn baby is at risk of genetic diseases.
Vertical farming: Soil-free agriculture in which plants are grown in vertically stacked layers or towers.
Hydroponics: Growing plants with their roots submerged in a nutrient-rich water solution instead of soil.
Aeroponics: Growing plants with roots suspended in air and sprayed with nutrient-rich water.
Aquaponics: Fish farming combined with hydroponics, forming a self-sustaining ecosystem.
Controlled environment agriculture (CEA): Technology (LED grow lights, temperature regulators and sensors) that maintains an optimal growing environment around the year.

Multiple choice questionsp. 208–209

Which tool of recombinant DNA technology is incorrectly paired with its use?

ARestriction enzyme: RFLP production
BDNA ligase: cuts DNAAnswer
CReverse transcriptase: makes cDNA
DElectrophoresis: separates DNA

Why

Restriction enzymes cut DNA at specific sites, so the fragments they produce differ in length between individuals (RFLP). DNA ligase does the opposite: it joins two DNA fragments by forming phosphodiester bonds, so 'DNA ligase: cuts DNA' is the wrong pairing.

Reverse transcriptase makes cDNA from mRNA (p. 191), and electrophoresis separates DNA fragments by size, so options A, C and D are all correct pairings.

Which of the following is a key difference between vertical farming and traditional agriculture?

AVertical farming uses more horizontal space, suitable for rural areas.
BVertical farming is not ideal for crops like wheat, rice, and sugarcane.
CVertical farming is ideal for urban areas; traditional farming needs large fields.Answer
DTraditional farming grows crops in vertical layers; vertical farming requires vast fields.

Why

Vertical farms stack plants in layers, so they use little horizontal space and suit cities where land is limited. Traditional farming needs large horizontal fields (Table 10.1, space utilization). Option A reverses this, and option D swaps the two systems.

Option B is also true, because vertical farms suit only short-cycle crops (Table 10.1, crop varieties). It describes crop choice, not the difference in land use that the question asks about.

A paleontologist finds tissue from a 400-year-old extinct bird and wants to compare its DNA with living birds. Which method would best increase the bird DNA for testing?

Aa RFLP analysis
Bpolymerase chain reaction (PCR)Answer
Celectroporation
DSouthern hybridization

Why

PCR makes thousands to millions of copies of a DNA segment in vitro, so the very small amount of DNA left in old tissue can be multiplied enough for testing (p. 198).

RFLP analysis and Southern hybridization detect and compare DNA fragments but do not increase their amount. Electroporation introduces DNA into cells; it does not copy DNA.

Which of the following sequences in double-stranded DNA is most likely to be recognized as a cutting site for a restriction enzyme?

A5'-AAGG-3' with 3'-TTCC-5'
B5'-AGTC-3' with 3'-TCAG-5'
C5'-GGCC-3' with 3'-CCGG-5'Answer
D5'-ACCA-3' with 3'-TGGT-5'

Why

Restriction sites are palindromic: the sequence reads the same 5' to 3' on both strands. Read the bottom strand 5' to 3' (right to left). Option C gives GGCC on the top strand and GGCC on the bottom strand, so it is palindromic.

The other options do not match. Option A's bottom strand reads CCTT, option B's reads GACT and option D's reads TGGT, and none equals its top strand. GGCC is the site cut by HaeIII (Fig. 10.3).

A plasmid

Ais used as DNA vectorAnswer
Bis a type of bacteriophage
Cis a type of cDNA
Dis a retrovirus

Why

A plasmid is a small, circular DNA molecule found naturally in bacteria that replicates independently of the chromosome. Because it has an origin of replication and can take up foreign DNA, it is used as a vector (p. 193).

A bacteriophage is a virus that infects bacteria, cDNA is made from mRNA, and a retrovirus is a virus with an RNA genome. None of these is a plasmid.

DNA molecules with complementary sticky ends associate by

Acovalent bond
Bhydrogen bondAnswer
Cionic bond
Ddisulphide bond

Why

Sticky ends are single-stranded overhangs whose bases are complementary. They pair through hydrogen bonds: A with T, and G with C.

The covalent phosphodiester bonds that join the backbone are formed afterwards by DNA ligase, so they are not what holds the ends together. Ionic and disulphide bonds do not hold DNA strands together.

Which technique rapidly replicates specific DNA fragments without cloning?

Agel electrophoresis
BcDNA libraries
Cgenetic probe
Dpolymerase chain reactionAnswer

Why

PCR copies a chosen DNA segment in vitro through repeated cycles of denaturation, annealing and extension. Millions of copies are made in a thermocycler without putting the DNA into a host cell (p. 198).

Gel electrophoresis separates fragments by size but does not copy them. A cDNA library and a genetic probe are used to find or detect DNA; they do not replicate a specific fragment.

The PCR technique uses

Aheat resistant DNA polymeraseAnswer
Breverse transcriptase
CDNA ligase
Drestriction enzymes

Why

PCR uses Taq polymerase, a heat-stable DNA polymerase isolated from Thermus aquaticus. It stays active at high temperatures, so it survives the repeated heating to 94 °C (p. 198).

An ordinary polymerase would be destroyed by the denaturation step. Reverse transcriptase makes DNA from RNA, DNA ligase joins fragments, and restriction enzymes cut DNA; none of them copies DNA in PCR.

If eukaryotic DNA contains five cleavage sites for a particular restriction enzyme, how many fragments will be produced upon complete digestion of the DNA with that enzyme?

A2
B4
C6Answer
DNone

Why

Eukaryotic chromosomal DNA is a linear molecule. One cut in a linear molecule gives two pieces, and each further cut adds one piece. So n cuts give n + 1 fragments: $5 + 1 = 6$ fragments.

If the DNA were circular, as a plasmid is, five cuts would give 5 fragments, so the answer depends on the DNA being linear, as eukaryotic chromosomes are.

The dideoxynucleotides (ddATP, ddTTP, ddGTP and ddCTP) are important in DNA sequencing because they:

Acause premature termination of growing DNA strandAnswer
Bare used as primers
Ccause the DNA fragments that contain them to migrate more slowly through the gel
Dare not affected by high temperatures

Why

A dideoxynucleotide has no 3'-OH group, so no further nucleotide can be added after it. When one is incorporated into a growing DNA strand, the strand stops growing (premature termination).

Option B is wrong because they are not primers. Option C is wrong because the shorter fragments that end in them move faster through the gel, not more slowly.

Note. The book prints 'are used as prime' for option B; it should read 'primers'.

Short answer questionsp. 209

13 marks

What is the role of restriction endonucleases in gene cloning?

Solution

Restriction endonucleases are molecular scissors. Each one recognizes a specific palindromic sequence (four to eight base pairs) and cleaves the phosphodiester bonds of both strands at that site. In gene cloning they do three jobs:

They cut the gene of interest out of the chromosomal DNA, so that a usable fragment is obtained.
They cut the plasmid vector at a site, so that the vector opens to receive the gene.
Because the gene and the vector are cut with the same enzyme, they carry complementary sticky ends that pair with each other, so the gene can be inserted in the right orientation.

23 marks

What are molecular carriers?

Solution

Molecular carriers, or vectors, are DNA molecules that carry a foreign gene into a host cell, where the gene can be copied and expressed. They act as a vehicle for the gene of interest. Plasmids are the most common example (p. 193). Other examples are lambda phage DNA, cosmids, yeast artificial chromosomes and bacterial artificial chromosomes.

A good vector has these properties:

an origin of replication, so that it copies itself in the host;
an antibiotic-resistance gene, as a selectable marker;
at least one restriction site, so the foreign DNA can be inserted;
a small size and high copy number, so that it is easy to insert and gives many copies.

33 marks

What is the role of restriction DNA ligases in gene technology?

Solution

DNA ligase is called molecular glue. It works after the restriction enzymes have cut the DNA:

It joins the sugar-phosphate backbones of the gene of interest and the vector by forming phosphodiester bonds between adjacent nucleotides (p. 195).
It seals the sticky ends that have paired by complementary base pairing, so the two fragments become one continuous recombinant plasmid.
It makes the join permanent, so the recombinant DNA is stable enough to be taken up by a host cell and replicated.

Note. The book prints 'restriction DNA ligases'; it should read 'DNA ligase', which joins DNA and does not cut it.

43 marks

What are the concerns about genetically modified organisms?

Solution

The main concerns about GMOs are safety concerns:

Waste and effluents: how to dispose of spent microbial biomass and purify the effluents from biotechnological processes.
Toxicity and allergy: the toxicity of, and the allergy associated with, the products of microbial production.
Antibiotic resistance: how to deal with the increase in antibiotic-resistant pathogenic microorganisms.

Other concerns are whether genetically engineered microorganisms can infect humans, plants and animals, and how to prevent contamination, infection or mutation of processed strains.

5(i)3 marks

Define biotechnology.

Solution

Biotechnology is the use of living organisms, especially microorganisms, or their processes and products, for the welfare of mankind (p. 190).

Examples: yogurt and cheese from fermented milk, wine or beer from fermented juices, and leather tanning by bacteria.
The term was introduced at the end of the 20th century.

5(ii)3 marks

Define genetic engineering.

Solution

Genetic engineering is the manipulation or alteration of the genetic material of an organism, by adding, removing or editing specific genes, to modify its characteristics or capabilities for human welfare (p. 190).

It is based on recombinant DNA technology, which combines DNA from different sources.
It is the branch of biotechnology that this chapter covers.

5(iii)3 marks

Define gene cloning.

Solution

Gene cloning is the production of many identical copies of a gene of interest by inserting it into a vector and multiplying it in a host cell, usually a bacterium.

The host multiplies the recombinant plasmid, so the gene is copied at industrial scale in culture tanks (p. 198).
The cloned gene can then be isolated for analysis, or its protein product separated.

5(iv)3 marks

Define palindromic sequence.

Solution

A palindromic sequence is a sequence of four to eight base pairs in DNA in which the nucleotides read the same in reverse order on both complementary strands (p. 192).

Restriction endonucleases recognize these sequences as their cutting sites.
Example: GAATTC reads the same 5' to 3' on both strands, and is cut by EcoRI.

5(v)3 marks

Define recombinant DNA technology.

Solution

Recombinant DNA technology is the joining of DNA segments from different sources to create new genetic combinations. It is the foundation of genetic engineering (p. 190).

The gene of interest is inserted into a vector.
The recombinant vector is transformed into a suitable host, which then multiplies it or expresses the gene.

5(vi)3 marks

Define DNA ligase.

Solution

DNA ligase is the enzyme that forms phosphodiester bonds between adjacent nucleotides, joining two double-stranded DNA fragments into one molecule (p. 195).

It is called molecular glue.
It joins the gene of interest to the plasmid at their complementary sticky ends.

5(vii)3 marks

Define polymerase chain reaction (PCR).

Solution

PCR is an in vitro technique that amplifies a single gene, or a DNA segment, into thousands to millions of copies by repeated cycles of heating and cooling (p. 198).

Each cycle has three steps: denaturation, primer annealing and extension.
It uses Taq polymerase and is carried out in a thermocycler.

5(viii)3 marks

Define primers.

Solution

Primers are short, single-stranded DNA sequences, made in the laboratory, that are complementary to the two ends of the target region on the template DNA.

They anneal to the template at about $55$ to $65^{\circ} C$ , and give Taq polymerase a 3' end from which to start adding nucleotides.
The forward and reverse primers anneal to opposite strands, so the region between them is copied (Fig. 10.8).

5(ix)3 marks

Define Taq polymerase.

Solution

Taq polymerase is a heat-stable DNA polymerase isolated from Thermus aquaticus, a bacterium of hot springs. It is stable and active at near-boiling temperatures (p. 198).

It synthesizes new DNA strands from the 3' end of each primer, using dNTPs, at about $72^{\circ} C$ .
Because it survives the repeated denaturation steps, it does not have to be added again in each cycle.

5(x)3 marks

Define genome.

Solution

The genome is the complete set of genetic material (DNA) of an organism. It contains all the genes that code for the organism's characteristics.

The genome is carried on the chromosomes; a chromosome is one of the DNA-carrying structures that make up the genome.
Example: the human genome includes the INS gene for insulin, which lies on chromosome 11 (p. 196).

5(xi)3 marks

Define transgenic organisms.

Solution

A transgenic organism is an organism that carries a foreign gene that has been deliberately inserted into its genome by genetic engineering (p. 203).

Transgenic bacteria, plants and animals have all been produced.
Transgenic animals that also produce human proteins or drugs are called transpharmer animals.

6(a)3 marks

Write the difference between biotechnology and genetic engineering.

Solution

Biotechnology is the broad field; genetic engineering is one branch of it.

Feature	Biotechnology	Genetic engineering
Scope	Broad field using living organisms and their processes for products and services	Narrow branch that changes the genetic material of an organism
Methods	Fermentation with microorganisms, for example yogurt, cheese and leather tanning	Recombinant DNA technology, restriction enzymes, vectors and host cells
Aim	Products and services for the welfare of mankind	Adding, removing or editing genes to give new or improved characteristics
Example	Wine or beer from fermented juices	Human insulin produced in bacteria

6(b)3 marks

Write the difference between staggered and blunt cut restriction enzymes.

Solution

The two types differ in where they cut the two strands of the DNA:

Feature	Staggered cut	Blunt cut
Cut in the two strands	The strands are cut at different points, so the ends overhang	Both strands are cut at the same point
Ends produced	Single-stranded sticky ends (5' or 3' overhangs)	Blunt ends with no overhang
Example	EcoRI (5' sticky ends); KpnI (3' sticky ends)	HaeIII
Use in cloning	Complementary sticky ends pair precisely, so the gene and vector join efficiently	Blunt ends can join to any blunt end, so the join is less precise and less efficient

Note. The book prints 'Kpn1'; the enzyme is KpnI.

6(c)3 marks

Write the difference between genome and chromosome.

Solution

The genome is the whole set of genetic material of an organism. A chromosome is one of the DNA-carrying structures that make up part of that genome.

Feature	Genome	Chromosome
Meaning	The complete set of DNA (genes) of an organism	One long DNA molecule, with proteins, carrying many genes
Relation	Includes all the chromosomes of the organism	A part of the genome
Example	The human genome, which contains the insulin gene	Human chromosome 11, which carries the INS gene (p. 196)

6(d)3 marks

Write the difference between transgenic organisms and hybrid organisms.

Solution

Both carry genes from more than one parent, but only transgenic organisms have a gene inserted deliberately by genetic engineering.

Feature	Transgenic organisms	Hybrid organisms
How produced	A gene from another organism is inserted by genetic engineering	Two genetically different parents are crossed by sexual reproduction
Genetic change	A foreign gene is added to the genome, often from a different species	Genes from both parents are mixed by normal inheritance; no DNA is inserted
Example	Bt cotton, which carries a gene from Bacillus thuringiensis	Offspring of two different varieties crossed by breeders

6(e)3 marks

Write the difference between biotechnology and nanotechnology.

Solution

Nanotechnology is not discussed in this chapter, so only a general difference is given:

Feature	Biotechnology	Nanotechnology
Material worked with	Living organisms, their cells and their processes	Matter at the scale of nanometres (about 1 to 100 nm)
Typical methods	Microorganisms, enzymes and genetic engineering	Designing and building materials and devices from individual molecules and atoms
Aim	Products for human welfare, such as medicines, food and industrial products	New materials and devices with special properties

Note. Nanotechnology lies outside this unit, so the answer gives a general definition.

73 marks

Shortly suggest, which type of farming is better from vertical farming and traditional agriculture farming in Pakistan.

Solution

Neither system is better in every case. The better choice depends on the crop and the place, so the best strategy is to use each where it suits:

Traditional agriculture is better for staple crops such as wheat, rice, sugarcane, cotton and maize. These need large fields, and traditional farming feeds the rural population.
Vertical farming is better in cities and in regions with water shortage, such as Thar and Baluchistan. It uses 90 to 95% less water, needs little land and gives higher yields of leafy greens and herbs all year.
Suggestion: traditional farming should remain the main source of staple food, while vertical farms supply fresh vegetables, herbs and fruit to urban areas with limited land.

83 marks

Explain the role of restriction endonucleases and DNA ligases in gene cloning.

Solution

The two enzymes do opposite jobs: one cuts the DNA and the other joins it.

Enzyme	Role in gene cloning	Bond acted on
Restriction endonuclease	Cuts the gene of interest and the vector at specific palindromic sites, producing matching sticky ends	Phosphodiester bonds are broken
DNA ligase	Joins the gene and the vector at their complementary sticky ends, sealing them into one recombinant plasmid	Phosphodiester bonds are formed

Cutting and joining with the same enzyme pair insert the gene of interest into the vector.

93 marks

Describe the selection and isolation of the gene of interest.

Solution

The gene of interest is the gene to be cloned. It can be obtained in one of three ways (p. 190):

Artificial gene synthesis: the gene is made in vitro, without a template, by a DNA synthesizer machine.
From mRNA: reverse transcriptase makes complementary DNA (cDNA) from the mRNA of the gene.
Cleavage of chromosomal DNA: restriction endonucleases cut the gene out of the chromosomal DNA. This is the most common method.

The gene is then isolated and purified so that it can be joined to a vector.

102026 Annual I, Set D, Q.2 (xi)3 marks

Explain the properties and the role of vectors in recombinant DNA technology.

Solution

A vector carries the gene of interest into a host cell, where the vector and the gene are copied and expressed. A suitable vector, such as pBR322, has these properties:

Origin of replication (ori): lets the vector copy itself in the host, so many copies of the gene are made.
Selectable marker: an antibiotic-resistance gene (for example ampicillin or tetracycline) that identifies the transformed cells.
Restriction sites: at least one site for the restriction endonuclease, so the foreign DNA can be inserted.
Promoter region: allows the inserted gene to be transcribed in the host cell.
Small size and high copy number: easy to purify and insert, and gives large amounts of the cloned DNA.

113 marks

State the steps for the integration of DNA insert into the vector.

Solution

The insert is joined to the vector in four steps:

Cut the vector with a restriction enzyme (for example EcoRI), so that the plasmid opens at one site and has sticky ends.
Cut the gene of interest with the same enzyme, so that its sticky ends are complementary to those of the vector.
Anneal the two DNA fragments: the complementary sticky ends pair by hydrogen bonds.
Ligate: add DNA ligase, which forms phosphodiester bonds and seals the fragments into one recombinant plasmid.

123 marks

Briefly state the technique applied for the selection of the vectors that take up the DNA insert.

Solution

The technique is antibiotic selection, which identifies the transformed clones:

The recombinant plasmids are introduced into bacteria, which are treated with calcium chloride to make them more permeable (transformation).
The bacteria are grown on a medium containing an antibiotic for which the plasmid carries a resistance gene, for example ampicillin.
Only the transformed bacteria survive and form colonies; the untransformed cells are killed by the antibiotic (p. 195).

133 marks

Describe the steps involved in gene amplification through polymerase chain reaction.

Solution

PCR amplifies a DNA segment in a thermocycler. Each cycle has three steps, and the cycle is repeated 25 to 35 times:

Denaturation at $94^{\circ} C$ for about 1 minute separates the double-stranded DNA into two single strands, which each act as a template.
Primer annealing at $55$ to $65^{\circ} C$ for about 2 minutes lets the forward and reverse primers bind to their complementary sequences on the single strands.
Extension at $72^{\circ} C$ for 1 to 2 minutes: Taq polymerase adds dNTPs to the 3' end of each primer and makes a new complementary strand.
Each cycle doubles the number of DNA copies, so the amount of product grows exponentially.

Before cycling, an initial denaturation at $94$ to $95^{\circ} C$ for 4 to 5 minutes is done. After the last cycle, a final extension at $72^{\circ} C$ for 5 to 7 minutes completes all the strands, and the product is held at $4^{\circ} C$ .

143 marks

State the objectives of the production of transgenic bacteria, transgenic plants and transgenic animals.

Solution

Transgenic organism	Objectives
Bacteria	Produce human proteins such as insulin; improve plant growth; remove environmental pollutants; extract metals from low-grade ores
Plants	Resistance to pests, diseases, herbicides and adverse environmental conditions; production of a nutrient or a pharmaceutical agent
Animals	Improve the traits of farm animals; produce drugs and human proteins (transpharmer animals)

153 marks

Explain the scope and importance of biotechnology in promoting human welfare.

Solution

Biotechnology is applied in four main fields (p. 190, p. 201):

Agriculture: genetically modified crops resistant to pests, diseases and environmental stress improve the food supply. Bt cotton has reduced pesticide use.
Medicine: insulin, antibiotics and vaccines are produced by genetic engineering, and gene therapy is researched for genetic disorders.
Industry: genetically engineered microorganisms help make biofuels, biodegrade industrial and sewage waste, and make environment-friendly chemicals.
Diagnosis: PCR detects infectious agents and genetic mutations, including those that cause genetic diseases and cancers.

163 marks

List the hazards of using genetically modified organisms.

Solution

The hazards of using GMOs are these:

Pollution from the spent microbial biomass and effluents, if they are not disposed of and purified.
Toxicity and allergy from the products of microbial production.
Spread of antibiotic-resistant pathogenic microorganisms.
Infection of humans, plants and animals by genetically engineered microorganisms.
Contamination, infection or mutation of processed strains.
Possible use of genetic manipulation for military purposes, which is a concern under the Biological Weapons Convention (1972).

Extensive answer questionsp. 209

110 marks

Describe the components of recombinant DNA technology under the following heads: (a) Gene of interest (b) Molecular scissors (c) Molecular carriers (d) Molecular glue (e) Expression system

Solution

Overview

Recombinant DNA technology has five components: the gene of interest, the molecular scissors (restriction endonucleases) that cut DNA, the molecular carrier (vector) that holds the gene, the molecular glue (DNA ligase) that joins the pieces, and the expression system (a host cell) in which the recombinant DNA is multiplied and expressed (p. 190).

(a) Gene of interest

The gene to be cloned is obtained in one of three ways:

Artificial gene synthesis in vitro, without a template, using a DNA synthesizer machine.
Synthesis from mRNA by reverse transcriptase, which makes complementary DNA (cDNA). This enzyme is found naturally in retroviruses.
Cleavage from chromosomal DNA by restriction endonucleases. This is the most common method.

(b) Molecular scissors

Restriction endonucleases cleave the phosphodiester bonds of both strands at specific palindromic sites. Bacteria use them as a defence against infecting viruses, and protect their own DNA by methylating its recognition sites (p. 191). Each enzyme is named after the genus, species and strain of the bacterium it comes from: EcoRI, for example, comes from Escherichia coli.

A staggered cut leaves single-stranded sticky ends (EcoRI), and a blunt cut leaves blunt ends (HaeIII). The steps of the EcoRI cut are shown in Fig. 10.2.

Figure 10.2: Recognition of a palindromic sequence by EcoRI and the production of sticky ends.

(c) Molecular carriers

A molecular carrier, or vector, carries the foreign DNA into the host cell. Plasmids are the common vector. pBR322 (4,361 bp) is the classic example: it has an origin of replication, two antibiotic-resistance genes (ampicillin and tetracycline) and several unique restriction sites, as shown in Fig. 10.4.

Figure 10.4: The E. coli cloning vector pBR322, with its origin of replication, antibiotic-resistance genes and restriction sites.

(d) Molecular glue

DNA ligase forms the phosphodiester bond between adjacent nucleotides. It joins the sugar-phosphate backbones of the gene and the vector at their complementary sticky ends, so it is called molecular glue. Without it, the annealed sticky ends would stay as two separate pieces held only by weak hydrogen bonds.

(e) Expression system

The expression system is the host cell or organism in which the recombinant DNA is multiplied (cloned) and expressed to make the protein. The selection depends on the vector. The most important feature of an ideal system is a short generation time and a simple genetic system, so bacteria, especially E. coli, are the usual choice (p. 195).

Component	Role	Example from the book
Gene of interest	The gene to be cloned	Human insulin (INS) gene
Molecular scissors	Cut DNA at specific palindromic sites	EcoRI, HaeIII
Molecular carrier	Carries the gene into the host and replicates it	Plasmid pBR322
Molecular glue	Joins the gene and the vector	DNA ligase
Expression system	Host that multiplies and expresses the gene	E. coli

28 marks

Describe the mechanism or procedure of recombinant DNA technology.

Solution

Overview

Recombinant DNA technology has four stages: formation of the recombinant plasmid, transformation of the host, identification of the transformed clone, and multiplication of the clone to obtain the product (p. 195).

Stage 1: Formation of the recombinant plasmid

Isolate the gene of interest and the vector (plasmid). The gene can be cut from chromosomal DNA or made from mRNA.
Cut both DNAs with the same restriction enzyme, so that they have compatible sticky ends (for example, EcoRI).
Mix them. The complementary sticky ends of the gene and the plasmid pair by hydrogen bonds.
Add DNA ligase. It forms phosphodiester bonds and seals the two fragments into the recombinant plasmid (recombinant vector).

Stage 2: Transformation

The recombinant plasmids are mixed with bacterial cells that contain no plasmids. The bacteria, made more permeable by calcium chloride, take up the plasmids. As they divide, a clone forms, and each new cell contains at least one plasmid carrying the gene of interest.

Stage 3: Identification of the transformed clone

The bacteria are grown on a medium containing an antibiotic for which the plasmid carries a resistance gene. Only the transformed cells survive and grow into colonies; the untransformed cells are killed. The transformed clone can then be used for further analysis.

Stage 4: Multiplication and product

The transformed clone is grown in large fermentation tanks. The plasmid is replicated in each cell, and the inserted gene is expressed to make its protein, which is then harvested and purified. In the insulin example, the chains are made as fusion proteins with beta-galactosidase (Fig. 10.6).

Figure 10.5: Steps for the formation of a recombinant plasmid, its transformation into a host cell and the selection of transformed clones.

32026 Annual I, Set B, Q.68 marks

What is polymerase chain reaction? Discuss its components and mechanism.

Solution

Definition

The polymerase chain reaction (PCR) is an in vitro technique that amplifies (copies) a specific DNA segment into thousands to millions of copies. It uses repeated cycles of heating and cooling, and needs no host cell (p. 198).

Components

Template DNA: the DNA segment to be amplified.
Primers: two short single-stranded DNA sequences, a forward and a reverse primer, complementary to the two ends of the target region.
dNTPs: deoxyribonucleoside triphosphates, the building blocks of the new DNA.
Taq polymerase: a heat-stable DNA polymerase isolated from Thermus aquaticus.
Buffer: a suitable buffer in which the components are diluted, placed in a thermocycler (PCR machine) that regulates the temperature and time of each step.

Mechanism

The reaction starts with an initial denaturation, then runs 25 to 35 cycles of three steps, and ends with a final extension and a hold at 4\,^\circ\mathrm{C}.

Initial denaturation: 94 to 95\,^\circ\mathrm{C} for 4 to 5 minutes completely separates the template DNA into single strands before cycling begins.
Denaturation (each cycle): 94\,^\circ\mathrm{C} for 1 minute separates the double-stranded DNA into two single strands, each of which acts as a template.
Primer annealing: 55 to 65\,^\circ\mathrm{C} for about 2 minutes. The forward and reverse primers bind to their complementary sequences on the single strands.
Extension (polymerization): 72\,^\circ\mathrm{C} for 1 to 2 minutes. Taq polymerase adds dNTPs to the 3' end of each primer, synthesizing a new complementary strand.
Each cycle converts every double-stranded molecule into two, so the number of copies doubles with each cycle.
Final extension: 72\,^\circ\mathrm{C} for 5 to 7 minutes completes any incomplete strands.
Storage: the product is held at 4\,^\circ\mathrm{C} until it is moved to a refrigerator.

Figure 10.8: Mechanism of one PCR cycle: denaturation at 94 °C, primer annealing at 55 to 65 °C and extension at 72 °C, repeated 25 to 35 times.

Stage	Temperature	Time	Purpose
Initial denaturation	94 to 95 °C	4 to 5 min	Separates the template completely
Denaturation	94 °C	1 min	Makes single-stranded templates
Annealing	55 to 65 °C	2 min	Primers bind to their target sequences
Extension	72 °C	1 to 2 min	Taq polymerase makes new strands
Final extension	72 °C	5 to 7 min	Completes all partly made strands

48 marks

Describe vertical agriculture farming. Explain hydroponics, aeroponics and aquaponics.

Solution

Vertical farming

Vertical (soil-free) farming grows plants without soil, in layers or towers stacked vertically, in a controlled environment. It is practised in urban areas, greenhouses, warehouses and small indoor spaces, where land is limited. The controlled environment uses LED grow lights with timers, temperature regulators and sensors to monitor growth (p. 205).

Because soil is heavy and would weigh down the stacks, the plants are grown in water or in air, with the nutrients supplied by the farm's system. On this basis the systems are called hydroponics, aeroponics or aquaponics.

1. Hydroponics

The roots of the plants are submerged in a nutrient-rich water solution. The water flows through channels, delivering nutrients directly to the roots, and is then recycled. Hydroponics uses up to 90% less water than traditional farming.

2. Aeroponics

The roots are suspended in air, and nutrient-rich water is sprayed directly onto them at specific intervals. The roots absorb moisture and nutrients efficiently and receive more oxygen. Aeroponics uses even less water than hydroponics.

3. Aquaponics

Fish farming is combined with hydroponics. Water from the fish tanks is circulated through the plant beds. The fish excreta provide natural nutrients to the plants, and in return the plants filter the water for the fish. The system is self-sustaining (Fig. 10.12).

Figure 10.12: Schematic of hydroponics, aeroponics and aquaponics, drawn from the descriptions in section 10.7.

System	Where the roots are	How nutrients reach the plant	Water use
Hydroponics	Submerged in nutrient solution	Solution flows through channels and is recycled	Up to 90% less than traditional farming
Aeroponics	Suspended in air	Nutrient-rich water is sprayed onto the roots at intervals	Less than hydroponics
Aquaponics	In plant beds	Fish excreta are the nutrient source, and the plants filter the water	Water is recycled between the fish tank and the plants

58 marks

Discuss the scope and importance of biotechnology in promoting human welfare.

Solution

Scope

Biotechnology uses living organisms and their processes to make products and services. Its scope covers agriculture, medicine, industry and the environment (p. 190).

Field	Applications
Agriculture	Genetically modified crops that resist pests, diseases and environmental stress; Bt cotton, which has reduced pesticide use; transgenic maize, soybean, cotton and canola
Medicine	Human insulin, antibiotics and vaccines; gene therapy for genetic disorders; PCR diagnosis of infectious agents and genetic mutations
Industry	Biofuels; biodegradation of industrial and sewage waste; environment-friendly chemicals; extraction of metals from low-grade ores by transgenic bacteria
Environment	Transgenic bacteria clean up environmental pollutants and increase the fertility of soil

Importance

Food security: genetically modified crops give higher yields and are protected against pests, so less of the crop is lost.
Health: insulin made by bacteria is purer and safer than insulin from animals, and PCR makes early diagnosis of disease possible.
Economy and environment: industrial processes, biofuels and waste treatment reduce pollution and the use of harmful chemicals.
Science: genetic engineering is one of the fastest-growing areas of science, and the 21st century has been called the 'Century of Biology' (p. 190).

68 marks

What are the ethical, legal and social implications of using biotechnology?

Solution

Ethical implications: safety

Waste and effluents from biotechnological processes must be disposed of and purified.
Microbial products may be toxic or cause allergy.
The increase in antibiotic-resistant pathogens must be controlled.
Engineered microorganisms must be evaluated for their ability to infect humans, plants and animals.
Processed strains must be kept free from contamination, infection or mutation (p. 202).

Social implications

Genetic counselling: trained counsellors explain the risks, benefits and limitations of genetic testing, and what the results mean for the person and the family (p. 203).
Genetic screening: it can affect parents' decisions about having children, and in some countries, such as Denmark, it is used in the approval of marriage licences (p. 203).
Large-scale transgenic crops: Bt cotton is grown on about 95% of Pakistan's cotton area, and GM sugarcane has been approved, so their safety must be assessed with care.

Legal implications

Biological Weapons Convention (1972): most countries have pledged never to produce microbial or other biological agents for use in wars. Concern remains that genetic manipulation could be used for military purposes (p. 202).
Approval before cultivation: new genetically modified varieties must be approved before they are grown, as GM sugarcane was in Pakistan (p. 202).

Type	Implication	Example from the book
Ethical	Safety of microbes and their products	Possible toxicity and allergy from microbial production
Legal	International commitment against misuse	Biological Weapons Convention, 1972
Legal	Approval of new GM varieties	GM sugarcane approved for cultivation in Pakistan
Social	Decisions by parents and society	Genetic screening used in approving marriage licences in Denmark
Social	Access to expert advice	Genetic counselling by trained healthcare professionals

78 marks

Compare vertical farming and traditional agriculture farming in Pakistan to clear advantages and disadvantages of both.

Solution

Comparison

The two systems differ on eight points. Table 10.1 compares them for each point.

Aspect	Vertical farming	Traditional agriculture
Space utilization	Advantage: stacks plants in layers and uses little horizontal space; ideal where land is limited or expensive	Disadvantage: needs large horizontal fields, which are hard to find in cities; wheat, rice and sugarcane need vast fields
Water usage	Advantage: uses 90 to 95% less water; hydroponics recycles water and aeroponics sprays it	Disadvantage: uses large amounts of water, especially for rice and sugarcane; floods and droughts waste water
Yield and productivity	Advantage: faster growth and higher yields per square foot, with year-round production	Disadvantage: depends on seasons and weather; floods, droughts and monsoons cause crop failures
Labour and automation	Advantage: automatic and AI systems reduce manual labour; disadvantage: high automation cost may be a barrier in rural areas	Disadvantage: depends on manual labour and outdated tools, so productivity is low
Crop varieties	Disadvantage: suits only short-cycle crops such as leafy greens, herbs and strawberries	Advantage: produces a wide range of crops, including grains, fruits and vegetables, which gives food security
Pesticides and chemicals	Advantage: few pests in controlled environments, so less pesticide and safer food	Disadvantage: frequent pesticide and fertilizer use, which can harm health and the environment
Environmental impact	Advantage: little soil erosion, less transport and little fossil fuel use; LED lights can use solar power	Disadvantage: at risk from salinity, floods and drought; deforestation to expand farmland
Accessibility and investment	Disadvantage: high initial investment, so less accessible to small farmers	Advantage: lower initial investment, suits small farmers; disadvantage: yields are low and unpredictable

Conclusion

Vertical farming has clear advantages in cities: it saves water, needs little land, avoids most pesticides and gives year-round yields of short-cycle vegetables. Its disadvantages are the high investment and its limit to short-cycle crops. Traditional agriculture remains essential for staple crops and for the rural population, because it grows the crops Pakistan depends on at low cost, although it is exposed to floods, droughts and high water use.

Note. The book prints 'to clear advantages and disadvantages'; it should read 'to state the advantages and disadvantages'.

FBISE 2026 questions

Questions from the FBISE HSSC-II 2026 (1st Annual) papers that belong to this unit, answered the way an examiner expects.

2026 Annual I, Set B, MCQ 111 mark

The first and the most important step in polymerase chain reaction:

ABlotting
BPrimer annealing
CPrimer extension
DDenaturationAnswer

Why

A PCR run begins with denaturation. Heat at 94 to 95 °C breaks the hydrogen bonds and separates the double-stranded template into single strands. Without single strands the primers cannot anneal and Taq polymerase has nothing to copy, so every later step depends on it.

B and C (primer annealing and primer extension) are the second and third steps of each cycle. A is wrong because blotting is not a step of PCR.

2026 Annual I, Set B, MCQ 151 mark

Plasmids are used as vectors because they:

ACannot carry foreign genes
BDestroy DNA of virus
CHave an origin of replicationAnswer
DOnly exist in viruses

Why

A plasmid has an origin of replication (ori), so it can copy itself independently of the chromosome in the host cell. This gives many copies of the gene inserted into it, which is what a vector must do.

A is wrong because a plasmid readily carries foreign genes once they are joined to it. B is wrong because a plasmid does not destroy viral DNA, and D is wrong because plasmids are found mainly in bacteria (and some yeasts and plants), not only in viruses.

2026 Annual I, Set B, Q.2 (xi) OR1+2 = 3 marks

a. What is meant by Genetically Modified Organism? b. Briefly state any two applications of Transgenic bacteria.

Solution

a. Genetically modified organism (1 mark)

A genetically modified organism (GMO) is an organism whose genetic material (DNA) has been altered in a way that does not occur naturally by fertilisation or natural recombination. Genetic engineering is used to insert foreign genes, remove existing genes or modify specific genes in it.

b. Two applications of transgenic bacteria (2 marks)

To produce human proteins, for example human insulin made in Escherichia coli (first done in 1978).
To improve plant growth.
To remove environmental pollutants.
To extract metals from low grade ores.

2026 Annual I, Set B, Q.65+1 = 6 marks

Describe the steps involved in Polymerase Chain Reaction PCR. Make flow sheet as well.

Solution

Definition

Polymerase chain reaction (PCR) is a technique that amplifies a single gene or piece of DNA into thousands to millions of copies by in vitro replication. It was invented by Kary Mullis in 1983 and is carried out in a thermocycler (PCR machine), which controls the time and temperature of each step.

Components of the reaction mixture

Template DNA: the DNA to be amplified.
Primers (forward and reverse): short pieces that mark where copying begins.
dNTPs: the free nucleotides, the building blocks of the new strands.
***Taq* polymerase:** a heat-stable DNA polymerase from Thermus aquaticus that synthesises the new strands.
Buffer: the medium of the reaction.

Steps (5 marks)

Initial denaturation (once): 94 to 95 °C for 4 to 5 minutes separates the template completely into single strands.
Denaturation (every cycle): 94 °C for 1 minute turns each double-stranded DNA into two single strands, and each strand acts as a template.
Primer annealing: 55 to 65 °C for about 2 minutes lets the forward primer (P1) and the reverse primer (P2) anneal to their complementary regions on the single strands.
Extension (polymerisation): 72 °C for 1 to 2 minutes, Taq polymerase adds nucleotides to the 3′ end of each primer and builds a new strand.
The three steps are repeated for 25 to 35 cycles. In each cycle the number of DNA molecules doubles, so the target sequence is amplified exponentially.
Final extension (once): 72 °C for 5 to 7 minutes completes any unfinished strands, and the product is stored at 4 °C.

Step	Temperature	Time	What happens
Initial denaturation	94 to 95 °C	4 to 5 min	Template fully separated
Denaturation	94 °C	1 min	Double strand becomes two single strands
Primer annealing	55 to 65 °C	About 2 min	Primers bind to the template
Extension	72 °C	1 to 2 min	Taq polymerase makes new strands
Final extension	72 °C	5 to 7 min	All strands completed
Storage	4 °C	Long time	Product kept safely

Flow sheet (1 mark)

Flow sheet of PCR: the three cyclic steps (accent border) are repeated 25 to 35 times between the initial denaturation and the final extension.

2026 Annual I, Set D, MCQ 121 mark

Polymerase chain reaction is a ______ technique.

ADNA degradation
BDNA hybridization
CDNA sequencing
DDNA amplificationAnswer

Why

PCR makes thousands to millions of copies of a chosen DNA segment in vitro, so it is a DNA amplification technique. The amount of DNA doubles with every cycle.

D is the only correct choice. B is tempting because primers hybridise to the template in the annealing step, but that is one step, not the aim of the technique. C is wrong because PCR only makes the DNA more abundant, which can simplify sequencing, but it does not read the sequence. A is wrong because nothing is degraded.

2026 Annual I, Set D, MCQ 161 mark

Plasmids are used as vectors because they:

AOnly exist in viruses
BCannot carry foreign genes
CThe destroy DNA of virus
DHave an origin of replicationAnswer

Why

A plasmid has an origin of replication (ori), so it replicates independently inside the host cell and makes many copies of the gene inserted into it. That is the property that makes it a vector.

A is wrong because plasmids are found mainly in bacteria, not only in viruses. B is wrong because a plasmid can carry foreign genes once they are joined to it. C is wrong because plasmids do not destroy viral DNA.

Note. The paper prints 'The destroy DNA of virus' in option C; it should read 'They destroy DNA of virus'.

2026 Annual I, Set D, Q.2 (xi)1+2 = 3 marks

What is plasmid? How plasmid acts as vectors in genetic engineering?

Solution

Plasmid (1 mark)

A plasmid is a small circular DNA molecule found naturally in bacterial cells. It lies outside the chromosome (extrachromosomal) and replicates independently.

Plasmid as a vector (2 marks)

A vector is a vehicle that carries foreign DNA (the gene of interest) into a host cell for multiplication and expression. A plasmid does this because the gene of interest is cut and joined into it with the same restriction enzyme and DNA ligase, and the recombinant plasmid is then put into a host bacterium. It is suitable because it has:

an origin of replication (ori), so it copies itself and the inserted gene many times (it is often present in high copy number);
an antibiotic resistance gene as a selectable marker, to find the bacteria that took up the plasmid;
unique restriction sites where the foreign DNA can be inserted;
a promoter, so that the inserted gene is expressed; and a small size, so it is easily purified and inserted into cells.

Main features of a plasmid used as a vector; the antibiotic resistance gene (accent) is the selectable marker.

2026 Annual I, Set D, Q.4 OR6 marks

Elaborate the steps of recombinant biotechnology taking the example of production of insulin.

Solution

Recombinant DNA technology joins the gene of interest to a vector, puts the recombinant DNA into a host and grows the host to make the product. Human insulin (two chains, A of 21 and B of 30 amino acids, joined by disulfide bonds) is made in Escherichia coli as follows.

Steps

Isolate the gene. The insulin gene (INS, on human chromosome 11) is cut from human DNA with a restriction endonuclease, or two synthetic genes, one for chain A and one for chain B, are made. E. coli cannot make a eukaryotic protein from the whole gene, so the two chains are made separately.
Select the vector. A plasmid such as pBR322 (or pUC18/19) is chosen. It has an origin of replication, high copy number, unique restriction sites and antibiotic resistance genes.
Form the recombinant plasmid. The plasmid is cut with the same restriction enzyme, so that its sticky ends are complementary to those of the gene. The sticky ends anneal and DNA ligase seals them with phosphodiester bonds. Each gene is placed next to the β-galactosidase gene, with a promoter, in a separate plasmid.
Transformation. The recombinant plasmids are put into E. coli (treated with calcium chloride so that it takes them up). The transformed cells are selected with the antibiotic: only bacteria that carry the plasmid's resistance gene survive.
Multiplication and expression. The transformed bacteria are grown in large fermentation tanks. The plasmid is copied and the genes are expressed, giving fusion proteins (β-galactosidase joined to chain A, and to chain B), which protect the chains from the bacterial proteases.
Extraction. The cells are harvested, and chains A and B are separated from the fusion protein by treatment with cyanogen bromide.
Joining the chains. Chains A and B are joined in vitro, with disulfide bridges between their cysteine residues, to give active insulin.
Purification, testing and packaging. The insulin is purified, tested for quality and safety, and packed in vials or pens. Recombinant human insulin is pure, safer and more effective than insulin from animals.

Production of human insulin in bacteria; the last step (accent border) gives the finished product.

Exam focus

Describe the steps of PCR with temperatures, components and a flow sheet. (long)
6-mark Set B Q.6; Set B MCQ 11 and Set D MCQ 12 ask the first step and the aim.
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Elaborate recombinant DNA technology with the production of human insulin as the example. (long)
6-mark Set D Q.4 OR; book long question 2.
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What is a plasmid and how does it act as a vector? List its features. (short)
Set D Q.2 (xi); Set B MCQ 15 and Set D MCQ 16 (origin of replication); book short question 24.
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Genetically modified organisms and the uses of transgenic bacteria, plants and animals. (short)
Set B Q.2 (xi) OR; book short question 28.
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Roles of restriction endonuclease and DNA ligase, and the selection of transformed clones by antibiotic. (short)
Unit's core tools; no direct paper question in 2026, so based on book short questions 1, 3, 22 and 26.
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Components and steps of PCR: why Taq polymerase is used and how the DNA doubles each cycle. (short)
Supports the 2026 PCR questions (Set B Q.6, MCQ 11, Set D MCQ 12); book short question 27 and exercise MCQ 7 and 8.
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