Genetic Technologies & Genomics

Genetic Engineering (GM)

Genetic engineering involves artificially altering an organism's DNA by transferring a gene from a donor to a host organism, creating a genetically modified organism (GMO).

1. Gene isolation: locate the desired gene
2. Cutting: use restriction enzymes to cut the gene from donor DNA and cut open a bacterial plasmid
3. Insertion: use ligase enzyme to splice the gene into the plasmid
4. Transformation: insert the recombinant plasmid into a host cell (e.g. bacterium)
5. Expression: the host cell expresses the new gene, producing the desired protein

Examples: human insulin produced by GM bacteria; Bt crops resistant to insects; Golden Rice with vitamin A.

DNA Profiling

Creates a unique genetic 'fingerprint' using non-coding DNA (which varies between individuals).

1. DNA extracted from sample
2. DNA amplified using PCR
3. DNA cut with restriction enzymes
4. Fragments separated by gel electrophoresis
5. Pattern of bands = DNA profile

Uses: forensic identification, paternity testing, solving crimes, identifying genetic disorders.

PCR (Polymerase Chain Reaction)

A technique to amplify (make millions of copies of) a small DNA sample. Uses cycles of heating and cooling with DNA polymerase.

Stem Cells

Stem cells are undifferentiated cells that can develop into many different cell types.

• Pluripotent: can become almost any cell type (embryonic stem cells)
• Multipotent: can become a limited range of cell types (adult stem cells)
• Therapeutic cloning: creating stem cells with the patient's own DNA to avoid immune rejection

Uses of Stem Cells

Treating diseases: Parkinson's, spinal cord injuries, diabetes. Bone marrow transplants use blood stem cells.

DNA Sequencing & Bioinformatics

DNA sequencing determines the order of nucleotide bases. Bioinformatics uses computers to analyse genomic data. Genome databases store DNA sequences and allow researchers to search for alleles linked to genetic diseases.

Ethical Issues

• Use of embryonic stem cells - destruction of embryos
• GM foods - environmental impact, consumer choice, labelling
• DNA databases - privacy, consent, data security
• Genetic screening - discrimination, insurance implications
• Gene editing (CRISPR) - designer babies, unintended consequences

PCR amplifies a specific region of DNA. It needs a DNA sample, two primers that mark the start and end of the target region, free nucleotides, Taq polymerase and buffer, all in a thermal cycler that changes temperature automatically.

One cycle, three steps

1. Denaturation (about 95°C): heat breaks the hydrogen bonds and the double helix separates into two single strands.

2. Annealing (about 55°C): cooling lets the primers bind to their complementary sequences at each end of the target region.

3. Extension (about 72°C): Taq polymerase adds complementary nucleotides from each primer, building a new strand.

Taq polymerase comes from Thermus aquaticus, a bacterium from hot springs, so it is not denatured at 95°C and does not need replacing each cycle.

Exponential amplification

Each cycle doubles the number of copies: 1, 2, 4, 8 and so on, so 2ⁿ copies after n cycles. Thirty cycles, taking a couple of hours, produce roughly a billion copies from a single starting molecule.

Applications

Forensic DNA profiling from a trace sample, diagnosing infections by detecting pathogen DNA (as in COVID-19 PCR tests), screening for inherited disease alleles, paternity testing, and preparing DNA for sequencing or genetic engineering. Because it is so sensitive, the smallest contamination can be amplified too, so negative controls and scrupulous technique are essential.

Bioinformatics is the use of computer software and databases to store, search and analyse biological data, especially DNA, RNA and protein sequences. A human genome is three billion bases long, so this analysis is impossible by hand.

Sequence alignment

Sequence alignment lines two or more sequences up base by base, or amino acid by amino acid, so that matches and differences can be counted. Software inserts gaps where bases have been inserted or deleted, and reports a percentage similarity.

What alignment tells us:

• Relatedness: the fewer the differences, the more recent the common ancestor, so alignments are used to build phylogenetic trees
• Mutations: comparing a patient's sequence with a reference sequence identifies the exact change causing an inherited condition
• Gene identification: an unknown sequence can be matched against known genes to suggest its function
• Tracking pathogens: aligning virus genomes from different patients shows how a variant arose and how an outbreak spread

Using a genome database

Public databases such as GenBank and Ensembl hold sequenced genomes, freely available worldwide. A typical search means choosing the organism, entering a gene name or pasting a sequence, running a search tool such as BLAST, and reading the results: matching species, percentage identity, gene location and known function. Results are ranked by how good the match is.

Open databases speed research because a scientist anywhere can compare a new sequence with everything already known, and personalised medicine uses the same approach to match a patient's genotype to the most effective drug. The limitations are real too: data quality varies, most sequenced genomes come from a narrow range of populations, and genetic data raises serious privacy and consent questions.

Genetic technology can now read, edit and store the information that defines an organism. The science tells us what is possible; ethics asks what we should do, and exam answers are expected to give balanced arguments on both sides.

Privacy and consent

A genome reveals disease risk not only for the person tested but for their relatives, who never consented. Who may see the data - insurers, employers, police? Direct-to-consumer testing companies hold huge databases that can be sold or breached, and children cannot consent to testing that will affect them as adults.

Human gene editing

Editing body cells to treat a disease affects only the patient. Editing embryos changes every future cell and is passed to descendants who cannot consent, and errors would be permanent. Most countries ban it, and there is a widely felt distinction between curing serious disease and selecting traits like height or intelligence, which raises the prospect of enhancement available only to the wealthy.

GM crops and animals

Arguments in favour: higher yields, less pesticide use, drought and pest resistance, added nutrients such as vitamin A in golden rice, and cheaper medicines such as insulin from GM bacteria. Arguments against: possible transfer of genes to wild relatives, effects on non-target insects, uncertainty about long-term effects, and the control that patents give a few large companies over farmers' seed. Welfare of genetically modified animals is a further concern.

Access and fairness

Gene therapies can cost a fortune, so who gets treated? Most genomic data comes from European populations, so results are less accurate for everyone else. Benefits and risks are unevenly shared between rich and poor countries.

How decisions are made

Ethics committees, national legislation and regulators weigh benefit against risk, require informed consent, insist on independent safety testing, and involve public consultation. Good scientific citizenship means understanding the evidence before forming a view.

Before DNA can be copied by PCR or separated by gel electrophoresis, it must be released from cells and separated from other cell material.

Method (e.g. from kiwi or onion)

1. Chop and blend the tissue briefly to break cell walls and separate cells.
2. Add washing-up liquid (detergent) to break down the lipid cell and nuclear membranes.
3. Add salt (sodium chloride) so DNA strands clump together and proteins separate.
4. Incubate in a water bath at 60 °C for 15 minutes to denature enzymes that would break down DNA. Do not go higher or the DNA itself breaks down.
5. Cool in an ice bath to slow any enzyme activity.
6. Filter to remove cell debris.
7. Add protease (e.g. from pineapple juice or contact-lens cleaner) to digest histone proteins wrapped around the DNA.
8. Pour ice-cold ethanol gently down the side of the tube. DNA is insoluble in cold ethanol and appears as white threads at the boundary. Spool it out with a glass rod.

DNA profiling (genetic fingerprinting) produces a pattern of bands unique to an individual, apart from identical twins. It does not read the whole genome. It looks at short repeating sequences in the non-coding DNA, where the number of repeats varies a lot from person to person.

The steps

1. Collect a sample - blood, saliva, hair root, semen or skin cells.
2. Extract the DNA from the cells: break the cell and nuclear membranes with detergent, add salt and protease, then add cold ethanol so the DNA precipitates out as white strands.
3. Amplify the chosen regions with PCR, so even a trace sample gives enough DNA to work with.
4. Cut the DNA into fragments using restriction enzymes, which cut at specific base sequences.
5. Separate the fragments by gel electrophoresis.
6. Compare the band patterns of the samples.

Gel electrophoresis

The DNA fragments are loaded into wells at one end of an agarose gel and a voltage is applied. DNA is negatively charged because of its phosphate groups, so all fragments move towards the positive electrode. Short fragments travel through the gel mesh faster and end up furthest from the wells; long fragments lag behind. A stain or fluorescent dye makes the bands visible. Two samples from the same person give identical band patterns.

Uses

Forensic investigation, identifying human remains, paternity and family testing, immigration cases, checking pedigree in animal breeding, detecting disease-causing alleles, and comparing species in evolutionary studies.

DNA sequencing and genome databases

Sequencing determines the actual order of bases in a piece of DNA. Fragments of every possible length are generated, each labelled according to its final base; separating them by size lets the sequence be read off in order. Modern high-throughput machines sequence a whole human genome in about a day, and the cost has collapsed from billions to a few hundred euro.

Sequences are deposited in public genome databases such as GenBank and Ensembl. Using one, you can choose an organism, search a gene or disease name, and find the alleles known to cause a condition, their chromosome location and their effect on the protein. This is how a diagnostic laboratory checks a patient's sequence against the reference to confirm a diagnosis.

Ethical issues

Who owns and controls genetic data? Could insurers or employers use it to discriminate? Should national DNA databases hold profiles of people never convicted? Consent, privacy and secure storage matter, and most sequenced genomes still come from a narrow range of populations, which limits how well the results apply to everyone.