Genetic Technology

A restriction endonuclease is an enzyme that cuts DNA at a specific point.

A recognition sequence is the specific short base sequence within the DNA at which the restriction enzyme cuts.

Recombinant DNA is DNA made by joining together DNA (sequences) from two different organisms / sources.

For example, a human gene (such as the insulin gene) inserted into a bacterial plasmid produces recombinant DNA.

A vector is something used to carry a gene (or DNA) into a host cell.

Transformation is the uptake of the vector / recombinant DNA by the host cell, so that the cell now contains the new gene.

A commonly used vector is a plasmid (a small circular piece of bacterial DNA).

First the desired human gene is isolated. The gene is then cut out using a restriction endonuclease, which cuts at a specific recognition sequence and leaves short single-stranded sticky ends (complementary overhangs).

The same restriction enzyme is used to cut open a plasmid (the vector), so that the plasmid has sticky ends that are complementary to those on the gene.

The cut gene and the cut plasmid are mixed. The gene's sticky ends base-pair (anneal) with the plasmid's complementary sticky ends by complementary base pairing. The enzyme DNA ligase then catalyses the formation of phosphodiester bonds in the sugar–phosphate backbone, sealing the gene into the plasmid to form a recombinant plasmid (recombinant DNA).

Finally, the recombinant plasmid is taken up by the bacterium (for example by heat shock with calcium ions, which makes the membrane more permeable). This uptake is called transformation; the transformed bacterium now contains and can express the human gene.

A marker gene is included in the plasmid alongside the gene of interest, so that transformed cells can be distinguished from untransformed ones.

If the marker is a gene for antibiotic resistance, the bacteria are grown on a medium containing that antibiotic. Only the bacteria that have taken up the plasmid possess the resistance gene, so only transformed bacteria survive and form colonies, while untransformed bacteria are killed. The surviving colonies are therefore the transformed cells.

Alternatively, a fluorescent marker such as the gene for green fluorescent protein (GFP) can be used: transformed cells glow under UV light, so they can be identified directly without killing the others.

In each case the marker is detectable only in cells that carry the plasmid, allowing the transformed bacteria to be selected.

A gene probe is a short, single-stranded length of DNA whose base sequence is complementary to the target disease allele, and which carries a label (radioactive or fluorescent).

The person's DNA is first made single-stranded (e.g. by heating) and is often amplified by PCR so there is enough to test. The labelled probe is then added.

The probe binds (hybridises) to the DNA only where it finds the complementary sequence, by complementary base pairing. The label is then detected — for example by autoradiography (radioactive label) or by fluorescence under UV. If a signal is found, the allele is present; if not, it is absent.

The probe must be single-stranded so that it can base-pair with the single-stranded target (a double-stranded probe could not bind). It must be labelled so that, after it has bound, its position can be detected — without a label there would be no way to tell whether the probe had bound.

A human gene cut directly from chromosomal DNA still contains introns (non-coding sequences) as well as the coding exons.

When this gene is transcribed in a bacterium, the bacterium cannot remove (splice out) the introns, because it lacks the splicing machinery found in eukaryotic cells. The introns are therefore translated along with the exons, so the protein produced has the wrong amino acid sequence and is non-functional.

Using cDNA solves this: cDNA is made by reverse transcriptase copying the cell's mature mRNA, which has already had its introns removed. The cDNA therefore contains only the exons (coding sequence), so when it is expressed in the bacterium the correct, functional protein is produced.

(a) Each PCR cycle has three stages:

• Denaturation (~95 °C): the hydrogen bonds between the bases break, separating the double helix into two single strands.
• Annealing (~50–60 °C): short single-stranded primers bind to each target sequence by complementary base pairing.
• Extension (~72 °C): heat-stable Taq DNA polymerase adds free nucleotides to each primer, building new complementary strands.

(b) Using $N = N_0 \times 2^n$ with $N_0 = 40$ and $n = 10$:

$N = 40 \times 2^{10} = 40 \times 1024 = 40\,960 \text{ copies}$

So there are 40 960 copies after 10 cycles.

Gene therapy delivers a working allele (often using a vector such as a modified virus or a liposome) to cells that carry a faulty allele, to treat a genetic disorder — for example delivering a normal CFTR allele to the lung cells of someone with cystic fibrosis.

Arguments in favour. Gene therapy could cure or relieve serious inherited diseases that currently have no cure, greatly improving quality of life. Somatic-cell gene therapy treats only the patient's body cells, so the change is not inherited; this is widely seen as acceptable because it only affects the individual being treated, who can give consent.

Arguments against. Germ-line gene therapy alters the gametes or embryo, so the change is passed on to all future generations. This raises serious concerns: the alteration is irreversible, it affects people not yet born who cannot consent, and unforeseen harmful effects would be inherited. There are also safety risks (for example a viral vector causing harm or inserting into the wrong place), and fears that the technology could be extended from treating disease to selecting desirable traits ('designer babies'), which many regard as morally wrong. For these reasons germ-line therapy is banned in humans in most countries.

Conclusion. On balance, somatic-cell gene therapy is ethically justifiable when used to treat serious disorders with the patient's consent, because the effects are limited to that individual. Germ-line therapy, however, should remain prohibited until its safety and the consent and 'designer baby' issues are resolved, because the heritable, irreversible changes affect future generations who cannot agree to them.

(a) There are strong arguments on both sides.

For: GM crops such as Bt crops can give higher yields (here a quoted 22% increase) and need less pesticide (a quoted 40% reduction), which lowers costs, reduces pesticide pollution and harm to non-target organisms, and can help feed a growing population. Crops can also be engineered to improve nutrition (e.g. golden rice making β-carotene to reduce vitamin A deficiency).

Against: there are concerns that introduced genes could spread to wild plants or other crops by cross-pollination, that growing a single modified variety could reduce biodiversity, and that pests may evolve resistance to the Bt toxin. Socially, GM seed is often controlled by a few large companies, which can disadvantage poorer farmers; some people also have moral or religious objections to altering organisms in this way, and worry about unknown long-term effects on health or the environment.

Conclusion: the clear yield and pesticide benefits make GM crops valuable, provided the risks are managed — for example by controlling gene escape, monitoring for resistance and ensuring fair access to seed. On balance the benefits can outweigh the risks if properly regulated, so a cautious, well-monitored use is justified.

(b) Before accepting that the GM crop caused the higher yield I would check: (1) whether the two crops were grown under the same conditions (soil, water, weather, fertiliser) so that the GM modification is the only variable; and (2) whether the trial was repeated over several plots/seasons with a large sample, rather than a single comparison, so the result is reliable and not due to chance or a one-off favourable patch — and whether the difference is statistically significant (correlation does not prove the gene caused the effect).