Genetic technology

📚The biology

The idea

Recombinant DNA is DNA formed by joining together DNA from two different organisms (or two different sources). Genetic engineering is the deliberate manipulation of genetic material to modify a specific characteristic of an organism, often by transferring a gene into it so that the gene is expressed. It is possible at all because the genetic code is universal — the same codon means the same amino acid in a bacterium as in a human, so a human gene put into a bacterium produces a human protein.

Getting the gene

Three routes, each with its own reason for being chosen:

• Extracted from donor DNA using a restriction endonuclease that cuts either side of the gene. Simple, but for a eukaryotic gene it brings the introns with it.
• Synthesised from mRNA using reverse transcriptase, which makes a complementary DNA (cDNA) copy. Because mature mRNA has already had its introns removed, the cDNA contains only exons — which is why this route is used when the host is a prokaryote, since bacteria cannot remove introns. You start from a cell that already makes the protein in quantity, so the mRNA is abundant.
• Synthesised chemically from nucleotides, once the sequence is known. Gives complete control over the sequence, including codons the host uses most efficiently.

The tools

• Restriction endonucleases cut DNA at specific base sequences (recognition sites). Many cut in a staggered way, leaving short single-stranded sticky ends. Cutting the gene and the plasmid with the same enzyme gives complementary sticky ends that base-pair with each other — that complementarity is the whole point.
• DNA ligase forms the phosphodiester bonds joining the sugar- phosphate backbones, sealing the gene permanently into the plasmid.
• Plasmids are small circular DNA molecules from bacteria, used as vectors to carry the gene into the host cell. They replicate independently of the bacterial chromosome, so the gene is copied every time the bacterium divides.
• DNA polymerase synthesises the second DNA strand from a cDNA template, and drives the polymerase chain reaction.
• Reverse transcriptase synthesises DNA from an RNA template.

Promoters and markers

A promoter is the sequence to which RNA polymerase binds to begin transcription. A gene without one that the host recognises will be inserted successfully and then never transcribed, so a suitable promoter must usually be transferred along with the gene. Promoters can also be chosen to control where and when the gene is expressed.

Only a minority of host cells take up the plasmid, so a marker gene is included to identify those that did. Modern practice uses a gene coding for a fluorescent product, such as green fluorescent protein: cells that have taken up the plasmid fluoresce under ultraviolet light and can be identified and separated directly. This is preferred to older antibiotic-resistance markers, which risked spreading resistance genes and required killing the non-transformed cells.

Gene editing is a form of genetic engineering involving the insertion, deletion or replacement of DNA at specific sites in the genome, rather than inserting a gene at a random position. That precision is its advantage: an existing faulty allele can be corrected in place.

The polymerase chain reaction

PCR amplifies a specific DNA sequence in vitro. Each cycle has three steps:

1. Denaturation, about 95 °C — the hydrogen bonds between the strands break, separating the DNA into two single strands.
2. Annealing, about 55–65 °C — short single-stranded primers bind to their complementary sequences either side of the target region, giving DNA polymerase a starting point and defining what is amplified.
3. Extension, about 72 °C — Taq polymerase adds free nucleotides to the primers, synthesising the complementary strands.

Taq polymerase comes from a bacterium living in hot springs and is thermostable: it is not denatured by the 95 °C denaturation step, so fresh enzyme need not be added each cycle and the whole process can be automated. That single property is what made PCR practical.

The quantity of DNA doubles each cycle, so \( n \) cycles from one molecule give \( 2^{n} \) copies — exponential amplification from a trace sample, which is why PCR underpins forensic science, diagnosis of infection, and ancient DNA work.

Gel electrophoresis

DNA fragments are placed in wells at one end of an agarose gel and a voltage applied. DNA is negatively charged because of its phosphate groups, so all fragments move towards the positive electrode (anode). The gel is a mesh: shorter fragments move faster and further, longer ones are held back. Fragments are therefore separated by length, and are visualised with a stain or a fluorescent or radioactive DNA probe. Comparing against a ladder of fragments of known length lets you estimate the size of each band.

Microarrays and databases

A microarray is a slide carrying thousands of different single-stranded DNA sequences at known positions. Labelled sample DNA or cDNA is washed over it and hybridises where it finds a complementary sequence; the pattern of labelled spots is read automatically. Used to compare genomes, and — when cDNA made from mRNA is used — to detect which genes are being expressed in a tissue, since mRNA is only present for genes being transcribed. Comparing a tumour with healthy tissue this way is a standard application.

Databases of nucleotide and amino acid sequences and protein structures let researchers anywhere compare sequences to identify genes, infer function from similarity to known proteins, establish evolutionary relationships, design primers and probes, and avoid repeating work — all without repeating the sequencing.

Medicine

Recombinant human proteins — the syllabus names three:

• Insulin — produced by genetically modified bacteria. It is identical to human insulin, so it works better and causes fewer allergic reactions than the animal insulin extracted from pigs and cattle that it replaced; it can be made in unlimited quantity; it is acceptable to people whose religion or ethics prohibit animal products; and there is no risk of transmitting animal disease.
• Factor VIII — for haemophilia. Previously extracted from donated blood, which carried a real risk of transmitting HIV and hepatitis; recombinant factor VIII removes that risk entirely and is not limited by donor supply.
• Adenosine deaminase — for a form of severe combined immunodeficiency.

Genetic screening tests for alleles associated with disease: BRCA1 and BRCA2 for breast cancer risk, HTT for Huntington’s disease, and the cystic fibrosis alleles. Advantages: early diagnosis and monitoring; informed reproductive decisions; preventive treatment or surgery; and identifying carriers.

Gene therapy inserts a functioning allele into a patient’s cells to treat a genetic disease. Named examples: SCID, and inherited eye diseases where the retina is accessible and the eye is immunologically privileged.

Social and ethical considerations — and note that a “discuss” question wants both sides:

• a positive result for an untreatable condition such as Huntington’s causes great anxiety and cannot be acted on medically;
• results may affect insurance and employment, raising the possibility of genetic discrimination;
• the result has implications for relatives who did not choose to be tested;
• confidentiality and informed consent are difficult when the information is shared by a family;
• gene therapy on somatic cells affects only the patient; therapy affecting germ cells would be inherited by future generations who cannot consent, and the long-term effects are unknown;
• the boundary between treating disease and enhancement is contested;
• treatments are expensive, raising questions of who receives them.

Agriculture

Genetic engineering can help meet the global demand for food by improving the quality and productivity of farmed animals and crop plants. Three named examples:

• GM salmon — carrying a growth hormone gene with a promoter that keeps it active year-round, so the fish reach market size in about half the time on less feed.
• Herbicide resistance in soybean — the crop tolerates a broad-spectrum herbicide, so weeds can be killed after the crop has emerged, reducing competition and simplifying cultivation.
• Insect resistance in cotton — a gene from the bacterium Bacillus thuringiensis makes the plant produce a protein toxic to the insect larvae that eat it, reducing crop loss and the need for insecticide spraying.

Ethical and social implications, both sides again: higher yields and better nutrition in food-insecure regions, and reduced pesticide use, against concerns about gene flow to wild relatives creating herbicide-tolerant weeds; the evolution of resistance in the target insects; effects on non-target species and on biodiversity; reduced genetic diversity in monocultures; the control of seed by a few large companies and the dependence of farmers on buying seed each year; long-term food safety, on which evidence to date is reassuring but limited; labelling and consumer choice; and unequal access between rich and poor countries.