What you'll learn
Genetic engineering and its applications covers the deliberate transfer of genes between organisms and the technologies that make it possible. At CAPE level you are expected to know the molecular tools in detail — restriction enzymes, ligase, vectors, markers — and to be able to describe the polymerase chain reaction, gel electrophoresis and DNA profiling, as well as to evaluate the ethical and ecological arguments that surround the field. By the end of this topic you should be able to describe how a gene is isolated, inserted and expressed, explain the function of each enzyme and vector, describe PCR and electrophoresis and DNA profiling, explain gene therapy, and construct a balanced evaluation of genetically modified organisms.
Key terms and definitions
Genetic engineering — the deliberate transfer of a gene from one organism to another, often of a different species
Recombinant DNA — DNA containing sequences from more than one organism
Restriction endonuclease — an enzyme that cuts DNA at a specific base sequence
Recognition site — the specific sequence at which a restriction enzyme cuts
Sticky ends — the short single-stranded overhangs produced by a staggered cut
DNA ligase — the enzyme that joins DNA fragments by forming phosphodiester bonds
Vector — the agent carrying the gene into the host cell, such as a plasmid or a virus
Plasmid — a small circular loop of DNA in bacteria, separate from the main chromosome
Marker gene — a gene inserted alongside the desired gene to identify transformed cells
Reverse transcriptase — the enzyme synthesising DNA from an RNA template
Complementary DNA — DNA made from messenger RNA by reverse transcriptase, abbreviated cDNA
Polymerase chain reaction — a technique for amplifying a specific DNA sequence, abbreviated PCR
Gel electrophoresis — separation of DNA fragments by size using an electric field
Gene therapy — the treatment of disease by introducing a functional allele into a patient's cells
Core concepts
Isolating the gene
Three routes are available, and the choice depends on the circumstances.
Restriction enzymes can cut the required gene out of the donor DNA. Each restriction endonuclease recognises a specific short base sequence and cuts the DNA there. Many cut in a staggered fashion, leaving short single-stranded overhangs called sticky ends, which are extremely useful because they will pair by complementary base pairing with any other fragment cut by the same enzyme.
Reverse transcriptase can synthesise the gene from messenger RNA. A cell actively expressing the gene is selected, since it will contain a high concentration of the relevant messenger RNA. Reverse transcriptase then uses that messenger RNA as a template to synthesise a complementary DNA strand, and DNA polymerase makes it double stranded. This method has a significant advantage for eukaryotic genes: because the messenger RNA has already been spliced, the resulting cDNA contains no introns and can therefore be expressed by a bacterium, which has no splicing machinery.
The gene can also be synthesised directly from the known base sequence using an automated gene synthesiser.
Inserting the gene into a vector
The most common vector is a bacterial plasmid.
The plasmid is cut open with the same restriction enzyme used to isolate the gene, so that its sticky ends are complementary to those of the gene.
The gene and the cut plasmid are mixed, and the complementary sticky ends pair by hydrogen bonding.
DNA ligase then joins the sugar-phosphate backbones by forming phosphodiester bonds, producing a closed circle of recombinant DNA.
Other vectors are used in different circumstances. Viruses, particularly bacteriophages for bacteria and modified viruses for human cells, insert their genetic material efficiently. Liposomes, spheres of phospholipid, can fuse with cell membranes and deliver DNA. Plant transformation frequently uses the bacterium Agrobacterium tumefaciens, which naturally transfers a section of plasmid DNA into plant cells.
Transformation and identifying success
The recombinant plasmids are mixed with host bacteria in a medium containing calcium ions, and the mixture is subjected to heat shock, which increases membrane permeability so that plasmids can enter.
The process is inefficient. Only a small proportion of bacteria take up a plasmid at all, and among the plasmids some will have closed up without incorporating the gene. Identifying the successfully transformed cells is therefore essential, and marker genes provide the means.
An antibiotic resistance marker works by including in the plasmid a gene conferring resistance to a particular antibiotic. The bacteria are grown on a medium containing that antibiotic, so only those that took up a plasmid survive.
Fluorescent markers are now more commonly used, since they avoid adding antibiotic resistance genes to the environment. A gene for green fluorescent protein, originally from a jellyfish, is inserted alongside the desired gene, and transformed cells fluoresce under ultraviolet light and can be identified without killing anything.
Enzyme markers work similarly: the marker gene produces an enzyme whose action on a substrate in the medium produces a visible colour change.
The transformed bacteria are then cultured in a fermenter and express the gene, producing the desired protein, which is extracted and purified.
The polymerase chain reaction
PCR amplifies a specific DNA sequence, producing millions of copies from a very small starting sample. It is automated and takes place in a thermal cycler that changes temperature in a repeating cycle of three stages.
Denaturation occurs at about 95 degrees Celsius. The high temperature breaks the hydrogen bonds between the base pairs, separating the double helix into two single strands.
Annealing occurs at about 55 to 65 degrees Celsius. Short single-stranded primers, complementary to the sequences flanking the region of interest, bind to the separated strands. The primers define which region is amplified and provide the starting point that DNA polymerase requires.
Extension occurs at about 72 degrees Celsius. DNA polymerase, together with free nucleotides, synthesises the complementary strand from each primer.
Each cycle doubles the number of copies, so after n cycles there are 2 to the power n times the original quantity. Thirty cycles therefore produce over a billion copies in a few hours.
The enzyme used is Taq polymerase, obtained from a bacterium living in hot springs. Its importance is that it is thermostable and is not denatured by the high temperature of the denaturation stage, so it does not need to be replaced each cycle. This single property is what made PCR practical, and it is regularly examined.
Gel electrophoresis and DNA profiling
Gel electrophoresis separates DNA fragments by size.
The DNA is cut into fragments with restriction enzymes and loaded into wells at one end of an agarose gel. A voltage is applied across the gel.
Because the phosphate groups of the sugar-phosphate backbone carry a negative charge, DNA fragments move towards the positive electrode. Smaller fragments move more easily through the pores of the gel and therefore travel further, while larger fragments are impeded and remain near the wells.
The fragments are visualised with a stain or by transferring them to a membrane and using a labelled DNA probe, which is a short single-stranded sequence complementary to the sequence of interest and carrying a radioactive or fluorescent label.
DNA profiling exploits the fact that non-coding regions of DNA contain short sequences repeated a variable number of times, and that the number of repeats differs between individuals. Restriction enzymes cut around these regions, PCR amplifies them, and electrophoresis separates them, producing a pattern of bands unique to the individual except in identical twins.
Applications include forensic identification, where a profile from a crime scene is compared with profiles from suspects; paternity testing, where a child's bands must each be present in one or other parent; and identifying genetic disease or assessing genetic diversity in conservation.
Applications of genetic engineering
Medically, bacteria have been engineered to produce human insulin, which avoids the former reliance on insulin extracted from cattle and pigs, produces a protein identical to the human form so that immune reactions are less likely, is available in unlimited quantity, and is acceptable to those with religious or ethical objections to animal products. Human growth hormone, factor VIII for haemophilia and vaccines are produced similarly.
Agriculturally, crops have been engineered for herbicide tolerance, allowing weeds to be controlled without damaging the crop; for insect resistance, by inserting a bacterial gene producing a toxin lethal to certain insect larvae; for improved nutritional content, as in rice engineered to produce beta-carotene to address vitamin A deficiency; and for tolerance of drought and salinity.
Gene therapy aims to treat genetic disease by supplying a functional allele. In somatic gene therapy the allele is introduced into body cells of the patient, so the treatment is not inherited and must usually be repeated as the treated cells are replaced. In germ line therapy the allele would be introduced into gametes or a very early embryo, so that all cells carried it and it would be passed to offspring; this is prohibited in most countries on ethical grounds.
Gene therapy has proved technically difficult. Delivering the allele to enough of the correct cells is problematic, the immune system may react to the viral vector, expression may be short-lived, and insertion at an inappropriate site in the genome risks disrupting another gene, which has in some trials caused cancer.
Evaluating genetic engineering
Examination questions frequently require a balanced evaluation, and both sides should be given with specific points rather than generalities.
The arguments in favour include increased crop yields and therefore food security; reduced pesticide use where insect resistance is engineered; crops able to grow in marginal conditions; production of pharmaceuticals in unlimited quantity and greater purity; and the potential to treat conditions with no other effective treatment.
The arguments against fall into three groups. Ecological concerns include the possibility of engineered genes spreading to wild relatives by cross-pollination, producing herbicide-tolerant weeds; harm to non-target species such as pollinating insects; and the selection of resistant pest populations. Health concerns include unknown long-term effects of consuming modified crops and the possibility of introducing allergens. Ethical and economic concerns include the patenting of seed by large companies, which may disadvantage small farmers; objections to transferring genes between species; and the question of who controls and benefits from the technology.
A strong answer concludes with a judgement referring to regulation, monitoring and case-by-case assessment rather than blanket approval or rejection.
Worked examples
Example 1: Explaining the use of the same restriction enzyme (4 marks)
Explain why the same restriction enzyme is used to cut both the donor DNA and the plasmid.
Each restriction endonuclease recognises and cuts at one specific base sequence, and many cut in a staggered fashion producing short single-stranded overhangs called sticky ends.
Because the same enzyme is used on both, the sticky ends on the isolated gene and on the opened plasmid have complementary base sequences.
The exposed bases can therefore pair with one another by complementary base pairing, forming hydrogen bonds and holding the gene in position within the plasmid.
DNA ligase then joins the sugar-phosphate backbones by forming phosphodiester bonds, producing a stable recombinant plasmid. If different enzymes were used, the ends would not be complementary and the gene could not be inserted.
Example 2: Explaining the advantage of cDNA (4 marks)
Explain why complementary DNA made using reverse transcriptase is often preferred to DNA cut directly from a eukaryotic chromosome when transferring a gene to a bacterium.
Eukaryotic genes contain introns, non-coding sequences that must be removed by splicing after transcription. Bacteria lack the enzymes to carry out splicing.
If the gene were cut directly from the chromosome, the bacterium would transcribe the introns along with the exons and would be unable to remove them, so the messenger RNA would be translated incorrectly and a non-functional protein would result.
Complementary DNA is synthesised from mature messenger RNA, from which the introns have already been spliced out. The cDNA therefore contains only the coding sequence, and the bacterium can transcribe and translate it correctly to produce a functional protein.
A further advantage is that messenger RNA for a particular protein is abundant in a cell specialised to produce it, so the gene is easier to obtain by this route.
Example 3: Interpreting a DNA profile (4 marks)
In a paternity case, the child's profile shows six bands. Four are present in the mother's profile. Of two possible fathers, man A shares the remaining two bands and man B shares neither. State the conclusion and explain the reasoning.
A child inherits half of its DNA from each biological parent, so every band in the child's profile must be present in the profile of one or other parent.
Four of the child's six bands match the mother's profile, so these were inherited from her. The remaining two bands must therefore have been inherited from the biological father.
Man A's profile contains both of these bands, so he could be the biological father. Man B's profile contains neither, so he cannot be the biological father and is excluded.
The evidence identifies man A as the probable father, though strictly it establishes that he cannot be excluded, and the strength of the conclusion depends on how many loci were examined.
Common mistakes and how to avoid them
The most frequent error is confusing the roles of restriction enzymes and ligase. Restriction enzymes cut; ligase joins.
Students often state that PCR uses ordinary DNA polymerase. Taq polymerase is used precisely because it is thermostable and survives the denaturation stage.
Another common slip is saying that smaller DNA fragments travel further because they are lighter. They travel further because they pass more easily through the pores of the gel.
Many candidates omit the marker gene from an account of genetic engineering, or fail to explain why it is needed. Transformation is inefficient, so identifying successful cells is essential.
In evaluation questions, answers frequently give only general statements such as that modified crops might be dangerous. Specific points — gene flow to wild relatives, allergen introduction, seed patenting — are what earn marks.
Finally, candidates often confuse somatic with germ line gene therapy. Somatic treats body cells and is not inherited; germ line would be inherited and is generally prohibited.
Exam technique for "Genetic engineering and its applications"
Describe the process as a numbered sequence — isolate, insert into vector, transform host, identify using marker, culture and express — since marks are allocated across the stages.
Name every enzyme and state its precise action. Restriction endonuclease, reverse transcriptase, DNA polymerase and ligase each carry marks.
For PCR, give the three stages with their temperatures and what happens at each, and explain why Taq polymerase is used.
In electrophoresis questions, state that DNA is negatively charged because of its phosphate groups and therefore moves to the positive electrode.
For evaluations, structure the answer into ecological, health and ethical or economic concerns, and finish with a judgement about regulation rather than a blanket verdict.
Quick revision summary
Genes are isolated by restriction endonucleases cutting at specific sequences to give sticky ends, by reverse transcriptase making intron-free complementary DNA from messenger RNA, or by direct synthesis. The gene is inserted into a vector, usually a plasmid cut with the same restriction enzyme so the sticky ends are complementary, and joined by DNA ligase forming phosphodiester bonds. Host bacteria take up plasmids after treatment with calcium ions and heat shock, and transformed cells are identified using antibiotic resistance, fluorescent or enzyme marker genes. PCR amplifies DNA through denaturation at 95 degrees, annealing of primers at 55 to 65 degrees and extension at 72 degrees by thermostable Taq polymerase, doubling the copies each cycle. Gel electrophoresis separates fragments by size, with negatively charged DNA moving to the positive electrode and smaller fragments travelling further. DNA profiling compares variable repeat regions for forensic, paternity and conservation purposes. Applications include human insulin, herbicide-tolerant and insect-resistant crops, and somatic gene therapy, while germ line therapy is generally prohibited. Evaluation should cover gene flow to wild relatives, effects on non-target species, unknown health effects, and the patenting of seed.