Genetic technology

Recombinant DNA and what genetic engineering is for

Recombinant DNA is DNA that has been formed by joining together DNA from two or more different sources. The usual arrangement is a gene taken from one organism inserted into a small circle of bacterial DNA, a plasmid, so that the gene can be carried into a host cell and copied there. Genetic engineering, also called genetic modification, is the deliberate manipulation of genetic material to modify specific characteristics of an organism, and the commonest form of it involves transferring a gene into an organism so that the gene is expressed and the organism makes a protein it could not make before. An organism that contains and expresses a gene from another species is described as transgenic.

The whole technology depends on one fact from nucleic acids and protein synthesis: the genetic code is universal and the machinery of transcription and translation works in almost the same way in every organism. A human gene placed in a bacterium is transcribed by bacterial RNA polymerase and translated on bacterial ribosomes into exactly the same polypeptide the human cell would have made. The gene is the instruction, and any cell that can read instructions can follow them - which is why a gene can be moved between species that last shared a common ancestor hundreds of millions of years ago.

The point of moving it is to get a useful product from an organism that is cheap and quick to grow, or to give an organism a characteristic that is useful to a patient or a farmer. Bacteria and yeast multiply rapidly in large fermenters, so a human gene for a protein such as insulin, factor VIII or adenosine deaminase can be turned into a medicine that is produced in quantity and is identical to the human protein. In agriculture the same approach gives a crop plant resistance to a herbicide or to an insect pest, and gives a farmed fish a faster growth rate.

Where the gene comes from is a decision with consequences. It may be extracted from the DNA of a donor organism, cut out with restriction endonucleases and identified among the fragments. It may be synthesised from the mRNA of a donor organism, because mRNA has already been spliced and so is a continuous coding sequence; the enzyme reverse transcriptase copies it into complementary DNA. Or it may be synthesised chemically from nucleotides, because an automated synthesiser can build a chosen sequence nucleotide by nucleotide from the known amino acid sequence of the protein.

Cutting, joining and delivering the gene: enzymes and plasmids

A restriction endonuclease is an enzyme that recognises a specific short sequence of bases, usually four to eight base pairs long, and cuts the sugar-phosphate backbone of the DNA at a particular point within that sequence. EcoRI, from Escherichia coli, cuts the sequence GAATTC. Many restriction enzymes cut the two strands at points that are not directly opposite each other, leaving short single-stranded overhangs called sticky ends. Bacteria use these enzymes as a defence against the viruses that infect them, cutting up viral DNA while protecting their own by adding methyl groups to their own recognition sites.

Sticky ends are what make neat gene transfer possible. The same restriction endonuclease is used on the donor DNA and on the plasmid, so the overhang at the end of the gene is complementary to the overhang on the opened plasmid and the two hydrogen bond together. DNA ligase then catalyses the formation of phosphodiester bonds between the sugar of one nucleotide and the phosphate of the next, sealing the gene into the plasmid so that the insert and the vector become one continuous DNA molecule. Restriction endonucleases cut and DNA ligase joins - swapping those two over is one of the commonest errors in this topic.

A plasmid is a small circular molecule of double-stranded DNA found in bacteria, separate from the main chromosome, and it is the standard vector, or carrier, for gene transfer. It is suitable because it has an origin of replication, so it is copied independently of the bacterial chromosome and the inserted gene is cloned as the bacterium divides; because it is small and circular and easily taken up; because it has recognition sites that a restriction enzyme can open; and because it normally carries a marker gene. Plasmids are put into bacteria by making the cell wall temporarily permeable to DNA, for example by treating the bacteria with calcium ions and a brief heat shock, or by electroporation. Other vectors are used where a plasmid will not do: bacteriophages and, for human cells, retroviruses and adeno-associated viruses; the Ti plasmid of Agrobacterium tumefaciens for plants; a gene gun that fires DNA-coated metal particles into cells; and direct microinjection into a fertilised egg.

Reverse transcriptase is the enzyme, found in retroviruses such as HIV, that makes a DNA copy from an RNA template - the reverse of transcription - and in genetic technology it is used to make cDNA from mRNA. DNA polymerase builds a new DNA strand by adding nucleotides to a free 3' end using the old strand as a template, and in the laboratory it is used to copy single-stranded DNA into double-stranded DNA, to label fragments as probes, and, in its heat-stable form, to amplify DNA in the polymerase chain reaction.

Promoters, marker genes and confirming that the gene is expressed

Cutting a gene out of donor DNA gives the coding sequence, but not the control sequences that switch it on. Transcription cannot begin unless RNA polymerase binds to a promoter, and the promoter that a human gene normally has is not recognised by a bacterium, so a promoter often has to be transferred into the organism as well as the desired gene. The promoter chosen also decides when and where the gene is expressed: a strong bacterial promoter gives a high yield in a fermenter, the CaMV 35S promoter from cauliflower mosaic virus drives strong expression in a plant, an inducible promoter can be switched on by adding a chemical to the culture, and a seed-specific promoter makes the gene work only in the seed.

Only a small proportion of the cells in a culture take up the plasmid, so the cells that have been transformed must be identified before they can be grown on. A marker gene is a second gene transferred at the same time, or built into the plasmid beforehand, whose product is easy to detect. Many marker genes code for a fluorescent protein, such as the green fluorescent protein (GFP) from the jellyfish Aequorea victoria: cells that have taken up the plasmid glow green when they are viewed under ultraviolet light, so they can be picked out and cultured.

Fluorescence is good evidence that the transferred gene is being expressed, not merely that the DNA has entered the cell. The fluorescent protein is only made if the transferred DNA has been transcribed and translated, so the glow shows that the host recognises the promoter, that transcription happened and that the mRNA was translated. That is why a fluorescent marker is used to confirm gene expression, and why the same reporter gene can be attached to the promoter of a gene being studied to show when and where that gene is switched on.

Antibiotic resistance genes are used in the same way. The plasmid carries a gene giving resistance to an antibiotic such as ampicillin, the bacteria are plated onto agar containing the antibiotic, and only the transformed cells survive and form colonies. The advantage of a fluorescent marker over an antibiotic resistance marker is that the cell is not killed, so the cells that glow can be separated one at a time and grown on. A marker gene does not prove that the protein is correct or that enough of it is being made, so the product itself is checked at the end of the process: the protein can be extracted and tested with an antibody that binds to it specifically, or the mixture of proteins can be separated by gel electrophoresis and stained so that the size and the amount of the product can be compared with a known sample.

• A promoter that the host recognises must be transferred with the gene, or the gene will not be transcribed and no protein will be made.
• Fluorescent markers: the transformed cells glow under ultraviolet light, so they can be identified and, if a cell sorter is used, separated individually.
• Antibiotic resistance markers: transformed cells grow on agar containing the antibiotic, untransformed cells do not.
• A reporter gene placed under the control of a promoter shows when and where a gene is switched on, because the fluorescent product is only made when that promoter is active.
• The gene may be taken up but not expressed, so identifying the DNA is not the same as confirming expression.

Gene editing: inserting, deleting or replacing DNA at a chosen site

Gene editing is a form of genetic engineering that involves the insertion, deletion or replacement of DNA at specific sites in the genome. The difference from ordinary gene transfer is precision and purpose: gene transfer adds an extra copy of a gene at a largely random position, whereas gene editing changes the existing sequence at a chosen place, which is what is needed to correct a faulty allele or to switch a gene off.

The system in general use is CRISPR-Cas9. A short guide RNA is made with a sequence complementary to the target site in the genome. The guide RNA base pairs with that site and the Cas9 endonuclease cuts both strands of the DNA there. The cell's own repair machinery then finishes the job in one of two ways. If the cut ends are simply joined back together, bases are usually lost or added at the join, so the gene is disrupted - an insertion or deletion that knocks the gene out. If a piece of donor DNA carrying the desired sequence is supplied at the same time, the cell can use it as a template during repair, so a faulty base sequence is replaced by a correct one.

CRISPR is precise because the guide RNA, about twenty bases long, decides exactly where the cut is made, and it is easy to redirect because changing the guide sequence is far simpler than redesigning a protein. Its natural role is part of the immune system of bacteria, which store short pieces of the DNA of viruses that have infected them and use them to recognise and cut the same DNA if it returns. Earlier editing tools worked in a similar way but targeted DNA through protein domains: zinc finger nucleases and TALENs, which are harder to design and more expensive to make.

Editing is already used in medicine and in research. Blood stem cells can be taken from a patient with sickle cell disease or beta-thalassaemia, edited so that they produce normal haemoglobin, and returned to the patient; a treatment of this kind, based on editing the gene that controls the switch from fetal to adult haemoglobin, was licensed for use in 2023. The technique is also used to knock out genes in laboratory organisms and cell lines so that their function can be worked out, and to make crops with a precise change rather than an added gene. The limits are that the enzyme can cut at a similar sequence somewhere else in the genome, producing an off-target edit, and that the change is confined to the cells that were treated. Editing the germ line, the cells that become eggs or sperm, would alter every cell of a future person and would be inherited by their children, who could not consent; it is not permitted in medicine, and the 2018 attempt to edit embryos to make them resistant to HIV was internationally condemned.

• Insertion: extra bases are added at the target site, which usually disrupts the gene.
• Deletion: bases are removed at the target site, so the reading frame or the coding sequence is changed.
• Replacement: a piece of donor DNA with the desired sequence is used as a template so that a faulty sequence is corrected.
• Targeting is by base pairing between the guide RNA and the target DNA, not by an enzyme that recognises a fixed sequence.
• Off-target cutting, the need to deliver the components to the right cells, and the ethics of germ-line editing are the main limitations.

PCR: cloning DNA in a tube

The polymerase chain reaction (PCR) makes millions of copies of a chosen length of DNA without using a living cell. The tube contains the DNA to be copied, a pair of primers (short single-stranded sequences, usually about twenty bases long, complementary to the sequence at each end of the target), free DNA nucleotides, a buffer containing magnesium ions that the enzyme needs, and a heat-stable DNA polymerase. The primers are what make the reaction specific: only the sequence between them is amplified, so a known flanking sequence is needed to design the reaction.

Each cycle has three temperatures, and you should be able to give the reason for each. Heating to about 95 °C denatures the DNA: the hydrogen bonds between the complementary bases break and the two strands separate. Cooling to about 55 °C lets the primers anneal, hydrogen bonding to their complementary sequences on each separated strand. Holding at about 72 °C allows extension: the DNA polymerase adds nucleotides to the free 3' end of each primer, building a new strand in the 5' to 3' direction, using the original strand as a template. The thermocycler then repeats the cycle, changing the temperature automatically.

Why the number of copies grows so quickly is worth being able to explain. Each cycle produces two new strands, one from each primer, and in the next cycle every strand - original or new - acts as a template. The number of copies of the target sequence therefore doubles each cycle: 2 after one cycle, 2 to the power of 20 (about one million) after twenty cycles, and more than a billion after thirty. That is why PCR matters practically: a single hair root, a drop of blood or a few cells from a swab contain enough DNA to be copied into a sample large enough to analyse.

The polymerase must survive the denaturation step, and the enzyme that does is Taq polymerase, from the thermophilic bacterium Thermus aquaticus, which lives in hot springs. Its tertiary structure is stable at 95 °C, so it is not denatured and does not have to be replaced after every cycle; it also has its optimum temperature close to the 72 °C used for extension. A human DNA polymerase would be denatured at 95 °C. Taq polymerase has no proofreading activity, so it inserts a wrong base more often than some other polymerases; where accuracy matters, a proofreading enzyme from a hyperthermophile such as Pyrococcus furiosus is used instead. Reverse transcriptase can be added to the same tube to copy RNA into DNA first, which is how RT-PCR detects RNA viruses.

Gel electrophoresis, microarrays and sequence databases

Gel electrophoresis separates DNA fragments by length. Every fragment carries a net negative charge, because its sugar-phosphate backbone has a phosphate group on each nucleotide, so in an electric field it moves towards the positive electrode (the anode). The gel is a mesh of agarose with pores through it: a short fragment slips through the pores easily and travels a long way in a set time, while a long fragment is held back and travels less far. The fragments are therefore sorted by size, measured in base pairs, and not by sequence. The DNA is loaded into wells at the negative end of the gel, a voltage is applied, the DNA is stained and the bands are viewed under ultraviolet light, with a sample of fragments of known length, a DNA ladder, run in one lane so that the length of each unknown band can be estimated from how far it has moved.

The technique becomes a DNA profile when the fragments come from repeated sequences that vary in length between people. Short tandem repeats are copied by PCR using primers that flank the repeat, so the product from each person is a set of fragments of characteristic lengths; the fragments are separated by electrophoresis and the pattern of bands, or of peaks from a capillary sequencer, is compared with a sample from a suspect or with a database. The method was developed by Alec Jeffreys at the University of Leicester, and its first use in a criminal case, in 1987, identified the man responsible for two murders in Leicestershire and also cleared an innocent suspect who had confessed. The same approach is used in paternity testing, in identifying remains and in checking whether a plasmid carries the insert it should.

A microarray is a small chip carrying thousands of short single-stranded DNA probes, each fixed at a known position. It is used in two ways. For analysis of genomes, DNA from a sample is labelled with a fluorescent dye and hybridised to the chip: fluorescence at a particular spot shows that the sample contains that sequence, and comparing a tumour sample with a normal sample shows which chromosome regions have been gained or lost. For studies of gene expression, mRNA is extracted from cells, reverse transcriptase makes labelled cDNA, and the cDNA hybridises to the probes; the amount of fluorescence at each spot shows how much mRNA of that gene was present, so a single chip shows which of thousands of genes are switched on. Comparing the pattern in a cancer cell with the pattern in a normal cell, or before and after a drug is added, identifies the genes involved in the response.

Databases of sequences make individual sequences meaningful. Nucleotide sequences of genes and genomes are held in GenBank at the National Center for Biotechnology Information and in the European Nucleotide Archive, and amino acid sequences and protein structures are held in UniProt and in the Protein Data Bank. A sequence determined in a laboratory can be compared with everything already known using a search tool such as BLAST, which identifies the gene, finds related genes in other species and allows the function of an unknown sequence to be inferred from a similar sequence whose function has been studied. Because the genetic code is known, a nucleotide sequence can be translated into an amino acid sequence, and an amino acid sequence can be used to predict how the protein folds, which is the starting point for designing a drug that fits it. Comparing whole genomes shows evolutionary relationships, and a variant found in a patient can be checked against reference sequences and databases of disease-causing variants to judge whether it is likely to cause the disease. The Human Genome Project, whose complete sequence was published in 2003, is the reference for this work. The limitations are that a database entry may be wrong, that a similar sequence does not prove a similar function, and that storing human sequence data raises questions of consent and privacy.

Genetic technology in medicine: recombinant proteins, screening and gene therapy

Recombinant human proteins have replaced proteins extracted from animal or human tissue, and the advantages are supply, safety and identity. Insulin for diabetes used to be extracted from the pancreas of pigs and cattle: the supply depended on the number of animals slaughtered, the product was slightly different in amino acid sequence from human insulin so some patients mounted an immune response to it, and the purification was slow and costly. Now the human insulin gene is transferred into a plasmid and expressed in Escherichia coli or in yeast grown in large fermenters, and the harvested protein is identical to human insulin, so it is available in unlimited quantity with consistent purity and no risk of animal disease. Factor VIII, the clotting protein that is missing or faulty in haemophilia A, used to be prepared from donated blood plasma pooled from many thousands of donors, and a single infected donation contaminated an entire batch; in the 1970s and 1980s many people with haemophilia were infected with HIV and with hepatitis C in this way, a tragedy examined recently by a public inquiry in the United Kingdom. Recombinant factor VIII, produced in cultured mammalian cells, carries no blood-borne virus. Adenosine deaminase is the enzyme missing in one form of severe combined immunodeficiency, and it can be supplied either as an injection of the recombinant enzyme or made by the patient's own corrected cells.

Genetic screening tests DNA for a faulty allele, and its advantages are that risk can be known before symptoms appear. Mutations in BRCA1 or BRCA2 greatly increase the risk of breast and ovarian cancer; a woman who tests positive can have more frequent imaging, can consider risk-reducing surgery, and may be offered drugs such as PARP inhibitors that are effective against tumours lacking these genes. Huntington's disease is caused by an expanded run of CAG triplets in the HTT gene and is inherited as an autosomal dominant condition, so a person with an affected parent has a 50% chance of carrying it; predictive testing tells them whether they will develop the disease, which allows them to plan their lives and their family. Cystic fibrosis is caused by mutations in the CFTR gene, and newborn screening by the heel-prick blood test, followed where necessary by a sweat test or a DNA test, means that a baby with the condition can start treatment early, which improves growth and lung function; carrier testing tells couples their chance of having an affected child.

Gene therapy treats a genetic disease by getting a working allele into the patient's cells. In severe combined immunodeficiency (SCID), in which no working immune system develops, the first human gene therapy trial took place in 1990: blood stem cells were removed from a child with ADA-SCID, a normal ADA gene was inserted using a retroviral vector, and the cells were returned to her, so that she made the missing enzyme herself. A later trial in Paris gave children with SCID-X1 a working copy of the gene, and it worked well, but some of the children later developed leukaemia because the vector inserted next to a gene that controls cell division - a reminder that a virus vector can disrupt the genome. Gene therapy has also been used in inherited eye diseases: in Leber congenital amaurosis caused by mutations in RPE65, a normal copy of the gene is injected under the retina using an adeno-associated virus vector, and some patients regain useful vision. The eye is a convenient organ because it is accessible, small and partly protected from the immune system, and trials for other inherited retinal diseases, including choroideraemia, have been run at Oxford.

Gene therapy and genetic screening raise ethical questions as well as medical ones. A test or a treatment can only be given with informed consent, and the information belongs to the patient, so confidentiality must be kept - a particular worry where insurers or employers might want the results, and the reason why insurers in the United Kingdom have agreed not to use predictive genetic test results. A result also belongs partly to other people: if one person is found to carry a faulty allele, their relatives may also carry it and may not want to know, and for a condition such as Huntington's disease, where knowing brings no cure, the effect on a person's mental health and on their decisions about children can be severe. Gene therapy has further problems of its own: vectors provoke immune reactions, insertions can disrupt genes, the effects are limited to the cells treated unless the germ line is changed, which is not permitted, and the licensed treatments cost very large sums, so they are out of reach in most health systems and raise the question of how a limited budget should be spent. Against those worries stand the relief of severe disease, the ability to plan a family, and treatment that is targeted at the cause of an illness rather than at its symptoms.

• Recombinant insulin: identical to human insulin, unlimited supply, no animal tissue and no immune response to an animal sequence.
• Recombinant factor VIII: no risk of transmitting HIV or hepatitis C from pooled donated plasma.
• Genetic screening: BRCA1 and BRCA2 for breast and ovarian cancer risk, the HTT gene for Huntington's disease, CFTR for cystic fibrosis and carrier status.
• Gene therapy: ADA-SCID by corrected blood stem cells, SCID-X1 by a retroviral vector, and RPE65 Leber congenital amaurosis by injection under the retina.
• Ethical issues: consent, confidentiality, insurance and employment discrimination, the effect of the result on relatives, germ-line editing, cost and unequal access.

Genetically modified organisms in agriculture

Genetic engineering can help meet the global demand for food by improving the productivity and quality of farmed animals and crop plants. GM salmon is the animal example. The AquAdvantage salmon is an Atlantic salmon carrying a growth hormone gene from Chinook salmon, together with a promoter from another fish, the ocean pout, that keeps the gene switched on all year instead of only in the warm season. Because the fish grows continuously rather than seasonally it reaches market size in a much shorter time, so a fish farm can produce the same quantity of food with less feed, less time and less space. The fish reviewed for approval were all female and triploid, so they are sterile, and they are raised in land-based tanks rather than in open sea cages, which reduces the risk that a modified fish could breed with wild salmon if it escaped. The species, the gene and the promoter are three separate facts, and an answer that muddles them loses marks.

In crop plants the two standard examples are herbicide resistance in soybean and insect resistance in cotton. Herbicide-resistant soybean carries a gene from a soil bacterium, Agrobacterium strain CP4, which codes for a form of the enzyme EPSP synthase that is not inhibited by the herbicide glyphosate. A farmer can spray the whole field and the crop survives while the weeds die, so weeds are controlled without cultivating the soil between the rows; the drawback is that repeated spraying of one herbicide selects for weeds that are also resistant, which has happened with pigweed in the United States. Insect-resistant cotton carries a gene from the bacterium Bacillus thuringiensis, coding for a protein (the Bt toxin) that is harmless to people, birds and most insects but binds to receptors in the gut of the larvae of certain pests, such as the cotton bollworm Helicoverpa armigera, and kills them. Because the plant makes the toxin itself, a farmer can grow a good crop with far fewer insecticide sprays, which saves money, protects the health of farm workers and reduces the number of harmless insects killed. Pests that are exposed to the toxin every season can evolve resistance to it, which is why growers are required to plant refuges of non-Bt plants nearby so that susceptible insects survive and mate with any resistant ones.

The use of genetically modified organisms in food production is opposed by some people and accepted by others, and the arguments are social and ethical as much as biological. Against: genes are moved between species that could never breed together, so the technology is seen as unnatural; genes for herbicide resistance can spread by pollen into wild relatives, producing weeds that cannot be sprayed; a patented seed must be bought again every season, which makes small farmers dependent on a few large companies; consumers want GM food to be labelled so that they can choose; and the long-term effects on biodiversity, on non-target insects and on the whole farming system are difficult to predict. For: there is no evidence from major scientific reviews that GM foods approved for sale are less safe to eat than conventional foods; insect-resistant crops reduce insecticide use and herbicide-resistant crops reduce ploughing, which cuts soil erosion and fuel use; Bt cotton has raised yields and reduced pesticide poisoning in the countries where it is grown; and varieties such as Golden rice, which makes beta-carotene in the grain and was developed to reduce vitamin A deficiency, are designed to improve nutrition for people who have the least. A good answer gives the biological mechanism behind the risk or the benefit, and then weighs the two sides against each other.

• GM salmon: a growth hormone gene from Chinook salmon with a year-round promoter from the ocean pout, raised in land-based tanks so that the fish cannot interbreed with wild salmon.
• Herbicide-resistant soybean: a bacterial EPSP synthase gene that survives glyphosate, so weeds can be killed without damaging the crop - but resistant weeds are selected for.
• Insect-resistant cotton: a Bt gene from Bacillus thuringiensis, so the plant makes a toxin that kills the larvae of the cotton bollworm and fewer sprays are needed.
• Golden rice: a crop engineered for a nutritional benefit rather than for yield, to reduce vitamin A deficiency.
• The two questions to keep apart are whether GM food is safe to eat and whether using it is fair, sustainable and acceptable.