Recombinant DNA technology

Three methods of obtaining a gene

The first requirement is a copy of the gene as DNA without introns, since a bacterial host cannot splice. Three methods are used, each suited to different circumstances.

Reverse transcriptase, working from mRNA. Find a cell that makes a great deal of the protein you want, such as the β cells of the pancreas for insulin or an immature red blood cell for globin, and it will be full of the corresponding mRNA. Extract that mRNA and treat it with reverse transcriptase, an enzyme obtained from retroviruses, which builds a DNA strand against an RNA template. The product is a single strand of complementary DNA, and DNA polymerase then builds the second strand against it, giving double-stranded DNA. Because the mRNA had already been spliced, the cDNA contains no introns and can be expressed in a bacterium.

Restriction endonucleases, working from the chromosome. If a suitable recognition sequence occurs on each side of the gene, an enzyme can cut it out directly. This is quick and needs no mRNA, but it delivers the gene complete with its introns, so the product is only useful in a host that can splice: a yeast or a mammalian cell culture, not E. coli.

The gene machine. The sequence is looked up in a database, or worked backwards from the amino acid sequence of the desired protein using the codon table, and then the DNA is built chemically, a nucleotide at a time, in short lengths that are joined by PCR into the whole gene. Nothing biological is required at all. The gene has no introns because none were put in, and the codons can be chosen from among the synonymous alternatives to suit the host organism's preferences, which can raise the yield of protein substantially. This is now how most genes are obtained commercially.

Reverse transcriptase

An enzyme, obtained from retroviruses, which catalyses the synthesis of DNA from an RNA template.

Complementary DNA (cDNA)

DNA made from an mRNA template by reverse transcriptase, and therefore containing no introns.

Recombinant DNA

DNA made by joining together sequences from two different organisms.

Transgenic organism

An organism containing DNA transferred into it from another species.

The technique depends on the genetic code being nearly universal: a bacterium translates the codon CCU as proline, as a human cell does. Human insulin has been manufactured in genetically modified bacteria since 1982, and before that diabetic patients used insulin extracted from the pancreases of pigs and cattle, which differs slightly in sequence and provoked immune reactions in some people.

Restriction enzymes, sticky ends and DNA ligase

Restriction endonucleases are bacterial enzymes that cut DNA at a specific sequence, usually four to eight base pairs long. Their function in the bacterium is defence: they hydrolyse the DNA of invading bacteriophages, and the bacterium protects its own genome by methylating it.

The recognition sequence is normally palindromic, meaning it reads the same 5′ to 3′ on both strands. EcoRI recognises GAATTC; the strand opposite reads GAATTC too, when read in its own direction. Some enzymes cut both strands at the same point, leaving blunt ends. The useful ones cut the two strands at different points, leaving each fragment with a short single-stranded overhang: a sticky end.

Describe the drawing

The recognition sequence of EcoRI, the six bases G A A T T C, written on the upper strand with its complement C T T A A G beneath it, so that both strands read the same way round. A bold line traces the cut, which falls between the G and the first A on the upper strand and at the mirror-image position on the lower one, so the two cuts are offset by four bases. Below, two separated fragments of DNA are drawn, one from a human chromosome and one from a plasmid, each carrying an exposed four-base overhang that reads A A T T, ready to pair with the other.

Sticky ends allow specific joining. Cut a human chromosome and a plasmid with the same enzyme and every fragment from both carries the same overhang, so the human fragment's exposed bases are complementary to the plasmid's. Mix them and the overhangs pair by hydrogen bonding. Those hydrogen bonds hold the two pieces in place but do not join the backbones, so the join is temporary until DNA ligase catalyses the formation of phosphodiester bonds along both strands. Name the two kinds of bond separately: hydrogen bonds between the paired overhangs, phosphodiester bonds along the backbones.

If no convenient site exists, the overhangs can be manufactured. Short single-stranded lengths of DNA carrying the wanted sequence are attached to the blunt ends of the fragment, and it then behaves as though it had been cut by that enzyme all along.

Vectors, transformation and the marker problem

A gene by itself in a bacterium is lost at the first division. It needs a vector: a piece of DNA that will be replicated by the host and passed to daughter cells. The usual choice is a plasmid, a small circular DNA molecule separate from the bacterial chromosome, carrying an origin of replication so the host copies it.

Four features are required on the plasmid besides the gene. An origin of replication, without which the plasmid is not copied. A promoter the host recognises, placed in front of the inserted gene: a human promoter is not recognised by E. coli RNA polymerase, so without a host promoter the gene is not transcribed. A terminator region behind it: without one, RNA polymerase continues past the end of the gene and transcribes the plasmid sequence beyond it, so the mRNA carries additional sequence and the yield falls. And a marker gene, for the reason given below.

Describe the drawing

A plasmid drawn as a large ring with three regions picked out along it in different colours: the gene of interest inserted at the top, a marker gene that the insertion has broken, and an origin of replication. A key to the right names each region and explains that placing the insert inside the marker is what makes the marker informative. Along the bottom, three linked panels describe mixing the plasmids with bacteria under calcium ions and heat shock, the very low proportion of cells that take a plasmid up, and the use of the marker to separate the populations afterwards.

Transformation is the uptake of the plasmid by the bacterium. The cells are made permeable: the standard method is to suspend them in an ice-cold calcium chloride solution and then give them a brief heat shock at about 42 °C; electroporation, a short high-voltage pulse, is the alternative. Either way the efficiency depends heavily on the protocol and is usually low: often only a small fraction of the cells take up a plasmid, and among the plasmids themselves many have re-closed on their own sticky ends without accepting an insert at all.

So after transformation the culture contains three populations: cells with no plasmid, cells with a plasmid that closed on itself, and cells with the recombinant plasmid you actually want. They look identical. The marker gene distinguishes them, and several designs are in use.

Antibiotic resistance markers are being replaced in commercial work, because handling large volumes of bacteria carrying resistance genes creates a risk of transfer to other bacteria. A fluorescent marker can replace the second, screening marker, but it cannot kill the cells that took up no plasmid, so some other selection is still needed first.

Getting DNA into other cells: viruses and the gene gun

Calcium chloride and a heat shock suit a bacterium. Other cells need other routes, and Pearson Edexcel Biology B names two of them: viruses and the gene gun. The method is chosen to suit the cell that has to receive the DNA.

Viral vectors. A virus is a delivery system already: it binds to receptors on a host cell and gets its nucleic acid inside. To make a vector, the genes the virus needs to replicate and cause disease are removed and the gene to be delivered is put in their place, so a particle can enter a cell and deliver its DNA but cannot make new viruses. Bacteriophages, the viruses of bacteria, are used to carry DNA into bacterial cells, and there the phage is usually left able to replicate, since that multiplies the inserted gene. For animal and human cells, retroviruses and lentiviruses insert the gene into a host chromosome, so it is copied at every later division, but at a random site; adenoviruses and adeno-associated viruses deliver DNA that mostly stays separate from the chromosomes and is gradually lost as the cells divide. Each virus infects particular cell types, which is useful when only one tissue is the target. These are the vectors gene therapy relies on.

The gene gun. A plant cell has a cellulose wall, and many crop plants, the cereals especially, were hard to transform by any other route. The gene gun, also called particle bombardment or biolistics, goes through the wall by force. Microscopic particles of gold or tungsten, about a micrometre across, are coated with copies of the DNA. A burst of high-pressure helium fires them at plant tissue, usually immature embryos or a callus of undifferentiated cells, fast enough to pass through cell walls and membranes. In a small fraction of the cells hit, some of the DNA reaches the nucleus and is inserted into a chromosome at a random position.

Those few cells have to be found and grown into whole plants. The construct carries a marker gene, often for resistance to an antibiotic or a herbicide, and the bombarded tissue is grown on a medium containing that substance, so only transformed cells survive. Plant cells are totipotent: a surviving cell, given the right balance of plant hormones, divides into a callus that then forms shoots and roots. Every cell of the regenerated plant carries the new gene, including the cells that will form its pollen and ovules, so the gene is passed on in seed. Particle bombardment also works where no biological vector does, on chloroplasts for instance. The commonest alternative for plants is Agrobacterium tumefaciens, a soil bacterium that naturally transfers part of its Ti plasmid into plant chromosomes.

Viral vector

A virus with its replication and disease-causing genes removed and a chosen gene inserted, used to carry that gene into host cells.

Gene gun

A device that fires microscopic gold or tungsten particles coated with DNA into cells, used chiefly to transform plant tissue.

The polymerase chain reaction, temperature by temperature

PCR produces many millions of copies of a chosen stretch of DNA in a few hours, in vitro. The cycle has three steps, each carried out at a different temperature.

The tube contains the DNA sample, a large excess of the four free nucleotides, two primers, and a heat-stable DNA polymerase in a buffer. The primers are short single strands of DNA, usually about twenty bases, complementary to the sequences at each end of the target region. They do two jobs: they give DNA polymerase the free 3′ end it requires to start, and they define which stretch of the sample is amplified. Different primers amplify a different region.

The specifications name two methods of amplification. PCR amplifies DNA in vitro: faster, requiring no cells, and yielding DNA only. Replication of a vector in host cells amplifies DNA in vivo: slower, but the culture can be scaled to industrial volumes. Protein is produced only when the insert is expressed, and correct folding and post-translational modification depend on the expression host. The choice follows from the purpose: a diagnostic test that requires enough DNA to sequence uses PCR; production of kilogram quantities of human insulin uses transformed cells in culture.

Describe the drawing

The polymerase chain reaction animated through one complete cycle, with the temperature of each step shown on a card at the left and the number of target molecules counted at the bottom. The tube starts with one double-stranded target, primers, Taq polymerase and free nucleotides. At 95 degrees the hydrogen bonds break and the two strands are drawn apart. At 60 degrees a short primer binds to one end of each separated strand. At 72 degrees, the optimum for Taq polymerase, a new strand is drawn growing from each primer. The last stage shows two complete double strands where there was one, and the count doubling with every further cycle.

Denaturation, 95 °C for about thirty seconds. Heat this high breaks the hydrogen bonds between the paired bases and the two strands separate. It does not break the phosphodiester bonds along the backbones, which are covalent, so each strand survives intact and can be used as a template. Nothing cooler will reliably separate a long double helix, particularly one rich in guanine and cytosine, where every pair is held by three hydrogen bonds instead of two.

Annealing, 50 to 65 °C for about thirty seconds. On cooling, the primers bind to their complementary sequences on the separated strands. The exact temperature is set by the primers themselves, by their length and their guanine-cytosine content, and 55 to 60 °C is typical. The temperature has to be set within a range. Above it, the primers do not remain bound, so no extension occurs. Below it, they bind to sequences they only partly match, and the reaction amplifies unintended regions.

Extension, 72 °C. This is the optimum temperature for Taq polymerase, the enzyme used, and it is why the step has a temperature of its own rather than simply staying at the annealing temperature. Taq extends each primer in the 5′ to 3′ direction at roughly a thousand bases a minute, so the length of this step is set by the length of the target. At 37 °C, the optimum for a human polymerase, Taq works far more slowly.

The cycle depends on Taq polymerase being thermostable. It comes from Thermus aquaticus, a bacterium of hot springs, and its tertiary structure is not disrupted at 95 °C. Before it was adopted, the polymerase was denatured at every denaturation step and fresh enzyme had to be added to every tube in every cycle, which made the technique impractical to automate.

copies after n cycles = 2ⁿ × starting moleculeswhich is exact only in principle: yield plateaus once the reagents start to run out

Gel electrophoresis: sorting fragments by length

Gel electrophoresis separates DNA fragments by length. It depends on the charge of the molecule: every nucleotide carries a phosphate group, and phosphate groups are negatively charged.

The samples are loaded into wells at one end of a slab of agarose gel covered in buffer, and a voltage is applied across it. Because all DNA is negatively charged, every fragment moves towards the positive electrode; charge does not distinguish them, since a longer fragment carries proportionately more charge. What distinguishes them is the gel. Agarose is a mesh, and a short fragment threads through it more easily than a long one, so in a given time the short fragments travel further. Distance moved falls roughly linearly with the logarithm of fragment length rather than with length itself, which is why size markers are run alongside.

Describe the drawing

A rectangular agarose gel drawn with four loading wells along its top edge and a long arrow down the left showing the direction the DNA travels, from the negative electrode at the wells towards the positive electrode at the far end. The first lane holds a ladder of five reference fragments of known length, 3000 down to 200 base pairs, each labelled at the right-hand edge. The other three lanes hold samples whose bands sit level with different rungs of that ladder, so their lengths can be read off directly.

DNA is colourless, so the gel has to be stained or the fragments labelled. Traditional practicals use methylene blue, which is safe but insensitive; research laboratories use fluorescent dyes that bind DNA and glow under ultraviolet light, or attach a fluorescent tag to a primer during the PCR that produced the fragments. Where the fragments have to be probed afterwards rather than merely measured, they are transferred from the gel onto a nylon membrane, a technique known as Southern blotting, and it is the membrane that meets the probe.

Knockout mice: switching a gene off to find its function

A genome sequence lists the genes but does not say what most of them do. One direct test is to stop a gene working in a whole animal and see what changes. A knockout mouse is a mouse in which both alleles of one chosen gene have been deliberately inactivated, so that no functional product is made. Comparing it with normal mice of the same strain shows what the gene contributes. If the inactivated gene is one that is faulty in a human disease, the mouse becomes an animal model of that disease, in which its course can be followed and treatments tested before any reach a patient.

The classic method, developed in the 1980s and recognised by a Nobel Prize in 2007, works through embryonic stem cells in five steps.

Gene editing has made the process much quicker. The Cas9 nuclease and a guide RNA matching the target gene are injected into a fertilised egg; the cut is repaired with small errors that disrupt the gene, and founder mice with the knockout can be obtained in one generation, though they are still bred to give a stable line.

Mice are used because they are mammals, with the same organ systems and much the same development and physiology as humans, and most human genes have a counterpart in the mouse genome. They are small and cheap to keep, breed at about two months old and have large litters. Inbred strains, in which the mice are almost genetically identical, mean that a difference between knockout and normal mice can be put down to the one gene that was changed.

Two examples show what the method gives and where it falls short. Mice lacking the tumour suppressor gene p53 are born apparently normal, but most develop tumours within months, which confirmed that the gene protects against cancer rather than being needed for development. Mice with the CFTR gene knocked out were made in 1992 as a model of cystic fibrosis. They suffer severe intestinal blockage but do not develop the human lung disease on their own, because mouse airways differ from human ones in the ion channels they carry.

Three further limitations recur in questions. A knockout may be lethal to the embryo, which shows the gene is essential but hides anything it does later in life; conditional knockouts, which switch the gene off in one tissue or at a chosen time, get round that. Another gene may take over the function, so the knockout looks normal although the gene does have a role. And a result in mice may not hold in humans, so a treatment that works in the model can fail in a trial. The animals may also suffer: in the UK every such experiment needs a licence under the Animals (Scientific Procedures) Act 1986, and researchers are expected to replace animals where they can, reduce the numbers used, and refine procedures to reduce suffering.

Knockout mouse

A mouse in which both alleles of a chosen gene have been deliberately inactivated, so that the gene's function can be inferred from what changes.

Animal model

An animal with a condition that resembles a human disease, used to study the disease and test treatments.

Evaluating genetic modification

Specifications require candidates to evaluate the ethical, financial and social issues raised by this technology. An evaluation sets out the evidence on both sides and reaches a stated judgement.

In agriculture. A crop engineered for pest resistance can cut insecticide use, raise yield on the same land, and carry nutrients a deficient diet lacks. Against that: the resistance gene may spread by pollen to wild relatives, pests evolve resistance in their turn exactly as they do to a sprayed insecticide, and a farmer paying for patented seed each season is in a different economic relationship from one saving it.

Three named organisms illustrate different aspects of the argument. Insect-resistant cotton carries a gene from the soil bacterium Bacillus thuringiensis for a protein toxic to the caterpillars that eat the crop, so the plant makes its own insecticide: spraying falls, yields rise, and the against-case is that constant exposure selects for resistant caterpillars faster than occasional spraying would, so refuges of non-GM cotton have to be planted alongside to slow it. Herbicide-resistant soybean carries a bacterial version of an enzyme that the herbicide glyphosate normally inhibits, so a field can be sprayed and only the weeds die: weed control is cheaper and ploughing can be reduced, and against that sit glyphosate-resistant weeds, heavier total herbicide use where they appear, and the patented-seed economics above. GM salmon makes the argument about animals: it carries a growth hormone gene from a chinook salmon under a promoter from another fish that runs all year rather than only in summer, so the fish reaches market size in roughly half the time on less feed. The case against is escape: a fast-growing transgenic fish breeding with wild populations could not be recalled. The approved fish are therefore reared as predominantly sterile triploid females in land-based facilities, with multiple containment measures. Check the current approval conditions for the jurisdiction named in a question, since they differ and are revised; the only commercial producer announced in late 2024 that it was stopping fish farming.

In industry and medicine. Human insulin from transformed bacteria is chemically identical to the human protein, avoids the immune reactions some patients had to insulin extracted from pigs and cattle, and can be produced in quantity and without the ethical objections that animal extraction carries. Against that: the development cost is high enough that research goes where the paying market is, which is not always where the disease burden is.

OCR Biology A names three further cases. Insect-resistant soya carries the same Bacillus thuringiensis toxin gene as Bt cotton, with the same arguments for and against. Genetically modified pathogens are made for research, for example strains with one gene knocked out to find what it does, or carrying a fluorescent marker so an infection can be followed; the benefit is understanding how a disease works, and the risk is escape or misuse, which is why the work is licensed and contained. Technology transfer asks whether patented GM seed reaches poor farmers: some has been licensed to them free of royalties, while patents and buying seed each season can keep it out of reach.

Structuring an evaluation. Attach each point to a named application rather than arguing about genetic modification in general, and separate the ethical, financial and social objections the command word names. State the mechanism behind an objection: "the resistance gene can transfer to a wild relative by pollen, and cannot then be recovered" identifies a process, whereas "it is unnatural" does not.

Genetically modified soya beans

Soya beans are grown for protein, for animal feed and for oil, and most of the soya grown in the Americas is genetically modified. Pearson Edexcel Biology B asks for the process and for two kinds of improvement: to the way the crop is grown, and to the oil itself.

The process follows the steps of this lesson. The gene is joined to a promoter that plant cells recognise, together with a marker gene. The construct is introduced into soya tissue with a gene gun or with Agrobacterium tumefaciens; the first herbicide-tolerant soya was made with a gene gun. Transformed cells are selected on a medium containing the herbicide or antibiotic that the marker protects against, and regenerated into whole plants. These are tested to confirm that the gene is present, expressed and inherited, and then crossed into high-yielding varieties suited to local conditions before the regulatory trials that any GM crop has to pass.

Improving production. Herbicide-tolerant soya, described in the section above, lets a farmer spray glyphosate over the growing crop: the weeds die and the soya does not, weed control is simpler and cheaper, and less ploughing is needed. Insect-resistant soya carrying a Bacillus thuringiensis toxin gene loses less of its crop to caterpillars. Both raise yield or cut costs, and neither changes the bean.

Changing the oil. Ordinary soya oil is rich in polyunsaturated fatty acids. About half of its fatty acid is linoleic acid, with two carbon-carbon double bonds, and about seven per cent is linolenic acid, with three. Oxygen attacks a fatty acid chain at the carbon atoms beside its double bonds, and the more double bonds a chain has, the faster it oxidises. Oxidised oil smells and tastes rancid, keeps poorly, and breaks down when it is heated repeatedly for frying. The traditional remedy was partial hydrogenation, adding hydrogen across some of the double bonds, but that process forms trans fatty acids, which raise the risk of heart disease and are now restricted in many countries.

Describe the drawing

A three-step pathway drawn left to right in a soya seed: oleic acid with one carbon-carbon double bond, converted by the enzyme FAD2 to linoleic acid with two, converted by FAD3 to linolenic acid with three. A coral cross on the FAD2 arrow marks the step silenced in high-oleic soya. Below each acid an amber bar shows its tendency to oxidise and turn rancid, shortest for oleic acid and tallest for linolenic acid, marked as relative and not to scale.

High-oleic soya deals with the problem in the seed. In the developing bean, an enzyme called fatty acid desaturase 2, FAD2, adds a second double bond to oleic acid, which has one, converting it to linoleic acid. In the genetically modified varieties FAD2 is silenced: an extra copy of part of the soya bean's own FAD2 gene is inserted, and its transcripts lead the cell to break down the normal FAD2 mRNA, a form of gene silencing related to the RNA interference met earlier in this unit. With little FAD2, oleic acid builds up instead of being converted. Over three-quarters of the fatty acid in the oil is then oleic acid, against roughly a fifth to a quarter in ordinary soya oil, with less linoleic and linolenic acid. The oil resists oxidation and stays stable in a fryer without hydrogenation, so no trans fatty acids are formed. A gene-edited high-oleic soya, with the FAD2 genes disrupted rather than silenced and no foreign DNA left in it, has been sold in the United States since 2019.

This modification improves the product rather than the farming. The benefit goes to food manufacturers and consumers, through longer shelf life and the removal of trans fats, rather than mainly to the grower, which is a different balance of benefit from the herbicide-tolerant crops at the centre of the public debate.

High-oleic soya

Soya genetically modified so that the enzyme converting oleic acid to linoleic acid is silenced, giving an oil with more monounsaturated fat that resists oxidation.