Genetic Engineering Techniques: cutting and joining DNA with restriction enzymes and ligase, transforming and cloning host cells, amplifying DNA by PCR, separating fragments by gel electrophoresis, and sequencing and genetic profiling

What this module is about

This is the toolkit at the heart of modern biotechnology. Having learned what DNA is, you now learn what to do with it: cut it precisely, join chosen pieces, deliver them into a host, copy a sequence millions of times, sort fragments by size, and read or compare the result. These five techniques recur throughout the applications modules, so a clear mental model of each is worth a great deal.

The full set of dot points for this module is at /sg-o-level/biotechnology/syllabus/genetic-engineering-techniques.

Cutting and joining: restriction enzymes and ligase

The dot point on restriction enzymes and ligase introduces the scissors and the glue. A restriction enzyme cuts DNA at a specific recognition sequence, often leaving short single-stranded sticky ends. DNA ligase joins pieces of DNA by sealing the sugar-phosphate backbone. The clever part is using the same restriction enzyme on both the gene and the plasmid: this produces matching sticky ends that pair by complementary base pairing, and ligase then seals them into a single recombinant DNA molecule.

Delivering and copying the cell: transformation and cloning

The dot point on gene cloning and transformation shows how the recombinant plasmid gets into a host and multiplies. The plasmid is mixed with host bacteria, which are treated (for example with calcium ions and a brief heat shock) so some take up the plasmid. Transformed cells are identified, often using a marker gene, then grown so each divides many times into a clone of identical cells all carrying the gene. This is how one engineered cell becomes a vat of product-makers.

Amplifying a sequence: PCR

The dot point on the polymerase chain reaction covers copying DNA outside a cell. PCR repeats a three-step cycle: denaturation (heat separates the strands), annealing (primers bind the target as it cools), and extension (a heat-stable DNA polymerase builds new strands). Each cycle roughly doubles the target, so the amount grows as a power of two.

Sorting by size: gel electrophoresis

The dot point on gel electrophoresis explains how to separate DNA fragments. Samples are loaded into wells in a gel and a current is applied. DNA is negatively charged, so fragments move towards the positive electrode, and smaller fragments move faster and further through the gel mesh than larger ones. The result is a pattern of bands sorted by size, read against a size marker. This is the read-out step for restriction analysis, PCR products and profiling alike.

Reading and comparing: sequencing and profiling

The dot point on DNA sequencing and genetic profiling draws an important distinction. Sequencing reads the exact order of bases in a length of DNA, for detailed study of genes. Genetic profiling (DNA fingerprinting) does not read every base; it compares highly variable regions to produce a banding pattern almost unique to an individual, used in identification and paternity testing. Both ultimately rely on the separation that gel electrophoresis provides.

How the module fits together

• Cut, then join. Restriction enzymes and ligase make recombinant DNA; the same-enzyme trick is the key idea.
• Deliver, then clone. Transformation gets the gene in; cloning multiplies the cells.
• Amplify, then read. PCR multiplies the sequence; electrophoresis and sequencing or profiling read it.
• Charge and size. Electrophoresis works because DNA is negative and small fragments travel furthest.

Check your knowledge

A mix of recall, reasoning and a calculation across the five dot points. Try them timed, then check the solutions.

1. State the function of a restriction enzyme and the function of DNA ligase. (2 marks)
2. Explain why the same restriction enzyme is used to cut both the gene and the plasmid. (2 marks)
3. Name the three steps of one PCR cycle in order. (2 marks)
4. In gel electrophoresis, towards which electrode does DNA move, and which fragments travel furthest? (2 marks)
5. State one difference between DNA sequencing and genetic profiling. (1 mark)