Changing one amino acid: site-directed mutagenesis

DNA carries the instructions for proteins in a simple code: each group of three bases, a codon, specifies one amino acid, and the same genetic code is read in non-overlapping triplets from a start codon (ATG) to a stop codon. Site-directed mutagenesis is the deliberate changing of a defined position in a cloned gene so that the effect of that one change can be studied: for example, replacing a single amino acid in a protein with alanine to test whether that residue is needed for the protein to work.

The method began with the demonstration that a short synthetic piece of DNA, matching a region of a genome but carrying a designed mismatch, could prime the synthesis of a new strand that bore a chosen mutation (Hutchison et al., 1978). Most present-day protocols use the same idea: a pair of complementary primers carrying the designed change is used to copy the whole plasmid by the polymerase chain reaction with a heat-stable, high-fidelity DNA polymerase (Saiki et al., 1988).

The plasmid being copied comes from an ordinary laboratory strain of Escherichia coli, which adds methyl groups to its DNA at GATC sequences (dam methylation). The enzyme DpnI cuts only this methylated DNA, so after the PCR it destroys the original, parental plasmid while leaving the newly made, unmethylated copies, which carry the mutation, untouched. The surviving DNA is then put into fresh E. coli, which repair the nicks and grow into colonies on an antibiotic plate, each colony a clone descended from a single molecule.

Because the polymerase copies the entire plasmid, it can occasionally introduce an unwanted change anywhere along it. Only the sequence of a clone shows that it carries the intended mutation and nothing else. The simulator records the true sequence of every clone, so the consequence of each choice, from primer design to the sequencing trace, is shown. The gene and plasmid are simulated and do not correspond to any real sequence.

Learning objectives

• Read a gene in codons, use the standard genetic code to translate it, and locate a chosen residue in the reading frame
• Choose an amino-acid substitution, work out the replacement codon from the codon table and judge how many base changes it needs
• Design a complementary mutagenic primer pair and check its length, melting temperature, GC content, flanking match and 3' end against the usual rules
• Explain why the whole plasmid is amplified, why a high-fidelity polymerase is used, and how DpnI removes the parental template
• Transform E. coli, select on an antibiotic plate, and interpret the colony numbers on the DpnI and no-DpnI plates
• Read a Sanger sequencing chromatogram to confirm the intended mutation and to detect unwanted second-site changes elsewhere in the gene

Ethics and safety

This work makes a genetically modified organism (E. coli carrying an engineered plasmid) and in a real laboratory requires prior approval by an Institutional Biosafety Committee (in India, under the Rules, 1989 of the Environment (Protection) Act 1986, with comparable national rules elsewhere) and must follow the containment and waste-disposal rules for the host strain. It is kept to standard, non-pathogenic laboratory strains of E. coli (K-12 type) and to harmless cloned genes; no pathogen or toxin genes are used. General laboratory safety training is needed: DNA stains and ultraviolet light used to view gels are hazards, so gloves and eye or face protection are worn and reagents are handled as the local risk assessment requires. Primers and constructs are recorded in full so that the work can be checked and repeated.

Key terms

• Plasmid: A small circular DNA molecule that replicates in bacteria independently of the chromosome. In the laboratory it is used as a vector to carry a cloned gene, usually together with an antibiotic-resistance gene for selection.
• Codon: A group of three DNA (or RNA) bases that specifies one amino acid, or a stop. The genetic code is read in non-overlapping triplets from the start codon.
• Reading frame: The way a sequence is divided into consecutive codons, set by the start codon (ATG). Adding or removing bases in numbers that are not a multiple of three shifts the frame.
• Open reading frame: A stretch of codons that runs from a start codon to a stop codon without an internal stop; the part of a gene that encodes the protein. Often abbreviated ORF.
• Point mutation: A change of a single base, or of a single codon, in the DNA. A missense change alters one amino acid; a silent (synonymous) change does not; a nonsense change creates a premature stop codon.
• Site-directed mutagenesis: The deliberate introduction of a chosen change at a defined position in a cloned DNA sequence, so that the effect of that one change can be studied.
• Mutagenic primer: A short piece of DNA, complementary to the target region but carrying the designed mismatch, that the polymerase uses as a starting point so the new strand bears the mutation.
• Melting temperature: The temperature at which half of a primer is bound to its complementary sequence. It rises with primer length and GC content and is used to judge how well a primer will anneal. Often written Tm.
• High-fidelity polymerase: A DNA polymerase with proofreading activity, which corrects most of its own copying errors, so fewer unwanted mutations are introduced when a whole plasmid is amplified.
• dam methylation: The addition of a methyl group to the A of every GATC sequence by the Dam enzyme of ordinary (dam+) E. coli strains. Plasmid DNA grown in such cells is methylated.
• DpnI: A restriction enzyme that cuts DNA only at methylated GATC sites. After mutagenesis by PCR it destroys the methylated parental plasmid and leaves the newly made, unmethylated product.
• Transformation: The uptake of DNA by bacteria made competent (permeable) for the purpose, so that a single plasmid molecule can enter a cell and be copied as the cell divides.
• Colony: A visible mound of bacteria on an agar plate, grown from a single founding cell, so that all its cells are genetically identical (a clone).
• Chromatogram: The trace produced by Sanger sequencing: a row of coloured peaks, one per base, read in order to give the DNA sequence and to reveal any change from the expected one.