Manipulating genomes: (ii) the techniques used in genetic engineering

The idea

Genetic engineering means deliberately transferring a gene from one organism into another so the recipient makes a new product. The core techniques form a sequence. A gene of interest is located and cut out using restriction enzymes that recognise specific palindromic sequences and leave sticky ends. A vector, usually a plasmid, is cut with the same enzyme so the ends are complementary. DNA ligase joins gene to vector to form a recombinant plasmid. The vector is introduced into a host cell, often a bacterium, by transformation, for example using calcium ions and heat shock or electroporation. Marker genes such as antibiotic-resistance genes allow transformed cells to be selected. Polymerase chain reaction can amplify the gene, and reverse transcriptase can make complementary DNA from mRNA. Finally the host is cultured and the product harvested.

What earns the marks

• Restriction endonucleases cut DNA at specific recognition sequences, producing sticky ends that allow complementary base pairing.
• The same restriction enzyme cuts the vector so that gene and vector ends are complementary and can anneal.
• DNA ligase catalyses formation of phosphodiester bonds, sealing the gene into the vector to form a recombinant DNA molecule.
• A vector such as a plasmid carries the gene into a host cell; transformation may use calcium ions and heat shock or electroporation.
• Marker genes, for example genes for antibiotic resistance, allow identification and selection of successfully transformed cells.
• The polymerase chain reaction amplifies DNA in vitro using primers, nucleotides, DNA polymerase and thermal cycling.
• Reverse transcriptase can synthesise complementary DNA from a mRNA template, giving a gene without introns.

Common mistakes and how to put them right

• Confusing restriction enzymes with DNA ligase.

  Put it right: Restriction enzymes cut, whereas ligase joins fragments by forming phosphodiester bonds.

• Assuming any enzyme can cut any vector.

  Put it right: The vector must be cut with the same restriction enzyme so the sticky ends are complementary.

• Thinking transformation is automatic.

  Put it right: Transformed cells must be selected, for example using a marker gene for antibiotic resistance.

• Believing PCR needs a host cell.

  Put it right: PCR amplifies DNA in vitro using thermal cycling, primers and DNA polymerase.

Exam tips

• Learn the techniques as an ordered workflow: cut, insert, transform, select, culture.
• Name the enzyme and its precise role each time, for example ligase forming phosphodiester bonds.
• Link each technique to its purpose, such as marker genes enabling selection of transformed cells.
• Use correct terminology: sticky ends, recombinant DNA, vector, transformation, complementary base pairing.