Question 1

What is the difference between Sanger sequencing and next-generation sequencing?

Accepted Answer

Sanger sequencing is a traditional method that uses chain-terminating dideoxynucleotides to sequence a single DNA fragment, typically up to 900 base pairs. It is accurate but slow and expensive for large genomes. Next-generation sequencing (NGS) technologies, such as Illumina sequencing, allow millions of fragments to be sequenced simultaneously in a massively parallel fashion, making it faster and cheaper for whole-genome sequencing. NGS uses reversible terminators and bridge amplification to read sequences in real time.

Question 2

How do restriction enzymes and DNA ligase work together in genetic engineering?

Accepted Answer

Restriction enzymes (restriction endonucleases) cut DNA at specific recognition sequences, often producing sticky ends (overhangs) that are complementary. DNA ligase then joins these sticky ends by catalysing the formation of phosphodiester bonds between the sugar-phosphate backbones. In genetic engineering, the same restriction enzyme is used to cut both the donor DNA (containing the gene of interest) and the vector DNA (e.g., plasmid), creating complementary sticky ends. DNA ligase then seals the gene into the vector, forming recombinant DNA.

Question 3

What is PCR and why is Taq polymerase used?

Accepted Answer

PCR (polymerase chain reaction) is a technique to amplify a specific DNA sequence, generating millions of copies. It involves repeated cycles of denaturation (heating to separate strands), annealing (cooling to allow primers to bind), and extension (heating to allow DNA polymerase to synthesise new strands). Taq polymerase is used because it is a thermostable enzyme isolated from the bacterium Thermus aquaticus, which lives in hot springs. It can withstand the high temperatures (around 95°C) needed for denaturation without denaturing itself, unlike human DNA polymerase.

Question 4

Can gene therapy cure genetic disorders permanently?

Accepted Answer

Somatic gene therapy can potentially cure a genetic disorder in the treated individual, but it is not always permanent. For example, in severe combined immunodeficiency (SCID), a functional copy of the defective gene is inserted into the patient's stem cells using a viral vector. These cells then produce the missing enzyme, but the effect may diminish over time if the modified cells die or are outcompeted. Additionally, there are risks such as insertional mutagenesis (the vector disrupting a tumour suppressor gene). Germ line gene therapy, which would affect future generations, is not currently permitted in humans due to ethical concerns and unknown long-term effects.

Question 5

What are the ethical concerns surrounding genetically modified (GM) crops?

Accepted Answer

Ethical concerns include potential environmental impacts, such as gene flow to wild relatives creating 'superweeds', and reduced biodiversity due to monoculture. There are also health concerns, though GM crops are rigorously tested and considered safe by major scientific bodies. Socially, there are issues of corporate control over seeds, patenting of life, and the impact on small farmers. On the other hand, GM crops can increase yields, reduce pesticide use, and enhance nutritional content (e.g., Golden Rice with vitamin A). A balanced view considers both the benefits and risks, and the need for regulation and public engagement.

Question 6

How does the Human Genome Project relate to manipulating genomes?

Accepted Answer

The Human Genome Project (HGP) was an international effort that sequenced the entire human genome, providing a reference map of all human genes. This knowledge is fundamental to manipulating genomes because it identifies the location and sequence of genes associated with diseases. For example, knowing the sequence of the CFTR gene allows scientists to develop gene therapies for cystic fibrosis. The HGP also spurred the development of faster sequencing technologies and bioinformatics tools, which are essential for modern genetic engineering and personalised medicine.

Manipulating genomes

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