Genetic technology

Every objective in this topic is printed under "A Level subject content" in the 9700 syllabus, so all of it is A Level and none of it is AS. It is examined on Paper 4. Paper 1 is the AS multiple-choice paper and does not reach this topic, so no multiple-choice practice on this site is tagged to it.

Syllabus points

19.1 Principles of genetic technology

• Define the term recombinant DNA.
• Explain that genetic engineering is the deliberate manipulation of genetic material to modify specific characteristics of an organism and that this may involve transferring a gene into an organism so that the gene is expressed.
• Explain that genes to be transferred into an organism may be: extracted from the DNA of a donor organism; synthesised from the mRNA of a donor organism; synthesised chemically from nucleotides.
• Explain the roles of restriction endonucleases, DNA ligase, plasmids, DNA polymerase and reverse transcriptase in the transfer of a gene into an organism.
• Explain why a promoter may have to be transferred into an organism as well as the desired gene.
• Explain how gene expression may be confirmed by the use of marker genes coding for fluorescent products.
• Explain that gene editing is a form of genetic engineering involving the insertion, deletion or replacement of DNA at specific sites in the genome.
• Describe and explain the steps involved in the polymerase chain reaction (PCR) to clone and amplify DNA, including the role of Taq polymerase.
• Describe and explain how gel electrophoresis is used to separate DNA fragments of different lengths.
• Outline how microarrays are used in the analysis of genomes and in detecting mRNA in studies of gene expression.
• Outline the benefits of using databases that provide information about nucleotide sequences of genes and genomes, and amino acid sequences of proteins and protein structures.

19.2 Genetic technology applied to medicine

• Explain the advantages of using recombinant human proteins to treat disease, using the examples insulin, factor VIII and adenosine deaminase.
• Outline the advantages of genetic screening, using the examples of breast cancer (BRCA1 and BRCA2), Huntington's disease and cystic fibrosis.
• Outline how genetic diseases can be treated with gene therapy, using the examples severe combined immunodeficiency (SCID) and inherited eye diseases.
• Discuss the social and ethical considerations of using genetic screening and gene therapy in medicine.

19.3 Genetically modified organisms in agriculture

• Explain that genetic engineering may help to solve the global demand for food by improving the quality and productivity of farmed animals and crop plants, using the examples of GM salmon, herbicide resistance in soybean and insect resistance in cotton.
• Discuss the ethical and social implications of using genetically modified organisms (GMOs) in food production.

Recombinant DNA and genetic engineering

Recombinant DNA is DNA made by joining together lengths of DNA from two different organisms, or from two different sources.

Genetic engineering is the deliberate manipulation of genetic material to modify the characteristics of an organism. Usually that means transferring a gene into an organism so that the gene is expressed, meaning transcribed and translated, so the organism makes the protein.

The whole field rests on one fact from topic 6: the genetic code is universal. The same triplet codes for the same amino acid in a bacterium, a plant and a human, so a human gene put into a bacterium produces a human protein. Without that, none of this would work.

An organism that has had a gene from another species inserted is transgenic, and it is a genetically modified organism (GMO).

Getting hold of the gene

Three routes are on the syllabus.

• Cut it out of the donor's DNA with a restriction endonuclease, provided the recognition sites fall in useful places.
• Synthesise it from mRNA. Take a cell that makes the protein in quantity, since it will be rich in the relevant mRNA. Beta cells of the pancreas are full of insulin mRNA. Use reverse transcriptase to make a single-stranded complementary DNA (cDNA) copy, then DNA polymerase to build the second strand. This route has a real advantage in eukaryotes: mRNA has already had its introns removed, and a bacterium cannot splice out introns, so cDNA is the only version a bacterium can express properly.
• Synthesise it chemically from nucleotides, once the base sequence is known from a database. This is now routine for short genes and lets the sequence be optimised for the host.

The enzymes and the vector

The reason sticky ends matter is worth stating rather than assuming. If the same restriction enzyme is used to cut both the donor DNA and the plasmid, both carry the same complementary overhangs, so the gene and the plasmid base-pair with each other by hydrogen bonding. Ligase then seals the joins permanently. Cutting with two different enzymes would give ends that do not match.

The plasmid is then taken up by a bacterium, usually after treatment with calcium ions and heat shock, or by electroporation, which makes the membrane temporarily permeable.

Why a promoter is needed too

A gene is only transcribed if RNA polymerase can bind upstream of it. The promoter is that binding site. A human gene inserted into a bacterium carries a human promoter, which bacterial RNA polymerase does not recognise, so the gene sits there and is never transcribed.

A promoter that the host recognises must therefore be transferred with the gene. Choosing the promoter also lets engineers control where and when the gene is expressed, which matters in a plant where you may want a protein in the seed but not in the leaf.

Marker genes

After transformation, only a small proportion of cells will have taken up the plasmid, and you need to identify them.

A marker gene is included in the plasmid alongside the gene of interest. Modern practice uses genes coding for fluorescent products, such as green fluorescent protein (GFP) from a jellyfish. Cells that have taken up the plasmid express GFP and glow green under ultraviolet light; cells that have not do not glow, and are discarded.

Because the marker is expressed only when the plasmid is present and being transcribed, fluorescence confirms not just uptake but expression. Fluorescent markers have largely replaced antibiotic-resistance markers, and the reason is a good exam point: releasing bacteria carrying antibiotic-resistance genes risks that resistance spreading to pathogens.

Gene editing

Gene editing is a form of genetic engineering in which DNA is inserted, deleted or replaced at a specific site in the genome, rather than a gene being added at a random position.

This is the important difference. Classical genetic engineering adds a gene wherever the vector happens to integrate, which may disrupt another gene. Editing targets a chosen sequence, so a faulty allele can be corrected in place, and the edited organism may contain no foreign DNA at all when the change is complete.

The polymerase chain reaction

PCR makes millions of copies of a specific length of DNA from a very small starting sample. One cycle has three steps, and it is repeated 25 to 35 times in a thermal cycler.

1. Denaturation, about 95 °C. The hydrogen bonds between the two strands break, so the DNA separates into single strands. The covalent bonds of the backbone are unaffected.
2. Annealing, about 55 to 65 °C. Short single-stranded primers base-pair with the ends of the target sequence. The primers define which stretch of DNA is amplified, and DNA polymerase cannot start without them.
3. Extension, about 72 °C. Taq polymerase adds free DNA nucleotides to the 3' end of each primer, building a complementary strand along each template.

Taq polymerase comes from Thermus aquaticus, a bacterium of hot springs. Its significance is that it is thermostable: it is not denatured by the 95 °C denaturation step, so it does not have to be replaced every cycle, and its optimum of about 72 °C sets the extension temperature. Before Taq, fresh enzyme had to be added by hand at every cycle.

The amount of DNA doubles each cycle, so the growth is exponential.

Worked example. One DNA molecule is put through 20 cycles.

number of molecules = 2²⁰ = 1048576

so a single molecule becomes over a million copies. After 30 cycles:

number of molecules = 2³⁰ = 1073741824

This sensitivity is what makes PCR useful in forensic work, in diagnosing infections from tiny samples, and in amplifying DNA from ancient remains. It is also why contamination is such a problem: a stray cell from the person handling the sample amplifies just as readily.

Gel electrophoresis

Electrophoresis separates DNA fragments by length.

1. Samples of DNA, usually cut with restriction enzymes, are placed in wells at one end of an agarose gel in a buffer solution.
2. A voltage is applied across the gel.
3. DNA is negatively charged, because of the phosphate groups in its backbone, so every fragment moves towards the positive electrode, the anode.
4. The gel is a mesh. Shorter fragments move further in a given time because they pass through the mesh more easily; longer fragments are held back.
5. The DNA is made visible by staining, or by using fluorescently labelled or radioactively labelled fragments and photographing the gel.

A DNA ladder of fragments of known length is run alongside, so the length of an unknown fragment can be read off by comparing how far it has travelled.

The key insight is that separation is by length only, since every fragment carries the same charge per unit length. That is why the charge on DNA never varies between fragments and cannot be the basis of separation.

Microarrays

A microarray is a slide carrying thousands of different single-stranded DNA probes, each of known sequence, fixed in a grid of spots.

• Genome analysis. Fragmented, fluorescently labelled DNA from a sample is washed over the array. Where a fragment is complementary to a probe it hybridises and stays bound; the rest is washed off. Scanning for fluorescence shows which sequences the sample contains, which is used to detect alleles associated with disease and to compare genomes between individuals or species.
• Gene expression studies. mRNA is extracted from cells and converted to labelled cDNA with reverse transcriptase. Only genes that were being transcribed produce mRNA, so the spots that fluoresce show which genes were active. Labelling two samples with different colours, for example healthy tissue and tumour tissue, shows at a glance which genes are more active in one than the other.

Bioinformatics databases

Sequence databases hold the nucleotide sequences of genes and genomes and the amino acid sequences and structures of proteins. Their benefits:

• Sequences can be compared between species, giving evidence of evolutionary relationships and identifying genes conserved because they matter.
• A newly sequenced gene can be searched against known sequences to suggest what its protein does, without any laboratory work.
• Primers for PCR and probes for microarrays can be designed straight from a published sequence.
• Predicted protein structures support the design of drugs that fit an active site.
• The data are shared and free, so results can be checked by other researchers and work is not duplicated.

Recombinant human proteins in medicine

The general advantages behind all three: large quantities can be produced, the protein is pure and exactly the human sequence so it works properly and provokes no immune response, and there is no risk of infection from a donor.

Genetic screening

Genetic screening tests an individual's DNA for particular alleles.

• BRCA1 and BRCA2. Mutations greatly raise the risk of breast and ovarian cancer. A woman who knows she carries one can have more frequent screening, take preventative drugs, or choose preventative surgery. The result is a risk, not a certainty, and that distinction matters.
• Huntington's disease. A dominant allele with late onset. A person with an affected parent can find out whether they carry it and use that in decisions about having children.
• Cystic fibrosis. Recessive, so screening identifies carriers who show no symptoms. Couples who are both carriers know there is a 1 in 4 chance for each child, and can consider IVF with embryo screening or prenatal testing. Newborn screening allows treatment to start before damage is done.

Advantages in general: earlier diagnosis and treatment, informed reproductive decisions, and identification of people who need monitoring.

Gene therapy

Gene therapy treats a genetic disease by inserting a functional allele into the patient's cells so the missing protein is made.

• SCID. In ADA-SCID, a faulty allele means no adenosine deaminase, toxic products accumulate and lymphocytes die, leaving the child with almost no immune system. Bone marrow stem cells are taken, a functional ADA allele is inserted using a viral vector, and the cells are returned. Because stem cells divide, the correction is passed to all the lymphocytes they produce, and the treatment can be long lasting.
• Inherited eye diseases. Certain forms of inherited blindness are caused by a faulty allele in the retina. A viral vector carrying the functional allele is injected directly into the eye. The eye is a good target because it is small, easily reached, and partly separated from the immune system, so a small dose can work and the response is less likely to be attacked.

Note the distinction between somatic gene therapy, which treats body cells and is not inherited, and germ line therapy, which alters gametes or embryos and would be passed to all future generations. Germ line therapy is illegal in most countries.

Social and ethical considerations

The syllabus asks you to discuss, which means arguments on both sides and a judgement.

For genetic screening: it allows early treatment and monitoring; it allows people to make informed decisions about their own lives and about having children; it can reduce the incidence of severe inherited disease; and knowing is often better than not knowing.

Against: results are often probabilities rather than certainties and are easily misunderstood; a positive result can cause severe anxiety, particularly for an untreatable condition such as Huntington's disease; there is a risk of discrimination by insurers or employers; screening embryos raises questions about which lives are considered worth having, and about how far parental choice should extend; and confidentiality is complicated by the fact that a result tells you about your relatives too.

For gene therapy: it treats the cause rather than the symptoms; it offers the only prospect for some otherwise fatal conditions; and somatic therapy affects only the patient, who has consented.

Against: the effect may be short-lived if the treated cells do not divide, so treatment must be repeated; viral vectors carry risks, including provoking an immune response and inserting the gene where it disrupts another, which has caused leukaemia in past trials; the cost is very high, which raises questions of who gets access; and germ line therapy would change future generations who cannot consent, and opens the way to enhancement rather than treatment.

Genetically modified organisms in agriculture

• GM salmon. A growth hormone gene from a Chinook salmon, with a promoter from another fish that keeps it active all year rather than seasonally, means the fish reaches market size in about half the usual time on less feed.
• Herbicide-resistant soybean. A gene giving resistance to a broad-spectrum herbicide lets a farmer spray the whole field, killing the weeds and leaving the crop. Weeds compete with the crop for light, water and mineral ions, so yields rise and less cultivation is needed.
• Insect-resistant cotton. A gene from the bacterium Bacillus thuringiensis makes the plant produce a protein toxic to the insect larvae that eat it. Crop losses fall and far less insecticide is sprayed.

Arguments for: higher yields from the same land, which matters as the population grows; less pesticide use and therefore less harm to non-target species; crops that tolerate drought or salinity can be grown where nothing grew before; and nutritional improvement, as with rice engineered to make beta-carotene.

Arguments against: genes may spread by pollen to wild relatives, creating herbicide-resistant weeds; insect populations may evolve resistance to the toxin, exactly as bacteria evolve antibiotic resistance; toxin-producing crops may harm non-target insects; farmers may become dependent on a single company for seed, which they cannot save and re-sow; long-term effects on human health are not fully known, though no harm has been demonstrated; and some object to moving genes between species in principle.

Common mistakes

• Defining recombinant DNA as any modified DNA, rather than DNA combining sequences from two different organisms.
• Saying restriction enzymes join DNA and ligase cuts it. Endonucleases cut; ligase joins.
• Cutting the plasmid and the gene with different restriction enzymes and still expecting the sticky ends to match.
• Forgetting the promoter, so the answer transfers a gene the host will never transcribe.
• Using genomic DNA rather than cDNA when the host is a bacterium, and so ignoring introns.
• Saying reverse transcriptase makes RNA from DNA. It makes DNA from RNA.
• Saying PCR needs Taq polymerase because it works fast. It is needed because it is not denatured at 95 °C.
• Leaving primers out of a PCR answer, or forgetting that the denaturation step breaks hydrogen bonds only.
• Saying long DNA fragments travel further in electrophoresis, or that fragments separate because they carry different charges.
• Saying DNA moves towards the negative electrode. It is negatively charged, so it moves to the positive one.
• Claiming somatic gene therapy is passed to a patient's children.
• Answering a "discuss" question with advantages only, and giving no judgement at the end.