Manipulating genomes: (f) (i) the principles of genetic engineering

The idea

Genetic engineering transfers a gene of interest from one organism into another so the recipient expresses the desired protein. The principles involve obtaining the gene, for example by PCR from DNA or by reverse transcription from mRNA, then inserting it into a vector such as a plasmid. The gene and vector are cut with the same restriction enzyme so the sticky ends are complementary and can be joined by DNA ligase. The vector is introduced into a host cell, often bacteria, and markers such as antibiotic-resistance genes allow transformed cells to be selected. Promoters and, in eukaryotes, appropriate control sequences ensure transcription and translation. The host then expresses the gene, producing the desired protein, which can be harvested.

What earns the marks

• The gene of interest is obtained, for example by PCR from DNA or by reverse transcription of mRNA to make cDNA.
• The gene and a vector such as a plasmid are cut with the same restriction enzyme, producing complementary sticky ends.
• DNA ligase joins the gene into the vector to form a recombinant DNA molecule.
• The vector is introduced into a host cell and transformed cells are identified using a marker such as an antibiotic-resistance gene.
• Control sequences such as a promoter allow the inserted gene to be transcribed and translated so the host expresses the desired protein.

Common mistakes and how to put them right

• Using different restriction enzymes for the gene and the vector.

  Put it right: The same enzyme is needed so the sticky ends are complementary and can be ligated.

• Confusing ligase with restriction enzymes.

  Put it right: Restriction enzymes cut DNA, whereas DNA ligase joins fragments.

• Assuming any host cell will express the gene without control sequences.

  Put it right: A suitable promoter and control sequences are needed for transcription and translation.

• Thinking selection markers create the gene.

  Put it right: Markers identify which cells have taken up the vector.

Exam tips

• Sequence the steps logically: obtain gene, cut gene and vector, ligate, transform host, select, express.
• Name the enzymes and state their specific roles rather than saying 'enzymes' generically.
• Link each stage to its purpose, such as complementary sticky ends enabling ligation.