DNA mutation and site-directed mutagenesis

Translation of a cloned gene, classification of mutations, and a defined point mutation taken through primer design, PCR, DpnI treatment, transformation and sequence-verified screening.

Guided experiment: Changing one amino acid: site-directed mutagenesis.

Site-directed mutagenesis is the deliberate alteration of a defined position in a cloned DNA sequence so that the effect of a chosen change can be studied. The approach began with the demonstration that a short synthetic oligonucleotide, complementary to a region of a viral genome but carrying a designed mismatch, could prime synthesis of a strand bearing a predetermined mutation (Hutchison et al., 1978). Most present-day protocols amplify the whole plasmid with mismatched primers using a thermostable DNA polymerase (Saiki et al., 1988), then remove the methylated parental template with DpnI before transformation. The simulator translates a cloned gene with the standard genetic code, classifies substitutions, insertions, deletions and codon edits, and takes a mutagenesis experiment from primer design to a confirmed clone. The gene and plasmid shown are simulated and do not correspond to any real sequence.

Design the experiment

Questions to investigate

Translate the gene and introduce a single-base substitution at different positions. When is the change silent, when missense, and when does it create a premature stop codon?
Insert or delete one base, then three bases. How does a frameshift differ from an in-frame change, and where does the new reading frame first meet a stop codon?
Choose a desired amino-acid substitution. Which replacement codon needs the fewest base changes, and can it be reached with a single-nucleotide change?
Design a complementary primer pair for the mutation. How do length and position of the mismatch affect the melting temperature, GC content and the risk of a hairpin?
Add a silent mutation that creates a restriction site. How does the diagnostic digest distinguish parental from mutant clones?
Compare high-fidelity and standard polymerase, and run with or without DpnI. How do the colony counts and the proportion of correct clones change, and why must the whole gene be sequenced?

In a real laboratory

The target gene is cloned in a plasmid with an antibiotic-resistance marker and maintained in a dam+ Escherichia coli host, so the parental DNA is methylated. Complementary mutagenic primers are designed, usually 25 to 45 nucleotides long, with the mismatch near the centre, at least about ten matched bases on each side, 40 to 60 per cent GC and a 3′ terminal G or C, and are checked for hairpins and self-complementarity. The whole plasmid is amplified with a high-fidelity polymerase, with a reaction without primers and an undigested reaction as controls. DpnI then cuts the methylated parental template but not the new, unmethylated strands, and the product is transformed and plated on selective medium. Colonies are screened by a diagnostic digest where a site was engineered, and every clone is sequenced across the entire gene, because the polymerase can introduce errors anywhere. A verified clone is stored as a glycerol stock with a record of its construction. Work that creates genetically modified organisms requires institutional biosafety approval.

How the simulation works

A seeded open reading frame of a few hundred codons, from ATG to a stop codon, lies in a simulated plasmid with a resistance marker. Translation uses the complete standard genetic code. Each edit is classified from the sequence: a substitution as synonymous, missense, nonsense, start-loss or stop-loss; an insertion or deletion as frameshift or in-frame, with the position of the first downstream stop codon and the length of the resulting protein. Primer melting temperature is approximated as Tm = 64.9 + 41 x (G + C - 16.4) / N for primers longer than 13 nucleotides, and hairpins are flagged by a simple stem search. Polymerase error rate, DpnI efficiency, transformation efficiency and colony numbers are approximate. Each picked clone is assigned at random to parental, correct mutant or mutant with a second-site change, and receives a simulated diagnostic digest and a Sanger read aligned to the reference. Codon usage, template secondary structure, real primer thermodynamics and the cell biology of transformation are not simulated.

Hutchison, C. A., Phillips, S., Edgell, M. H., Gillam, S., Jahnke, P., & Smith, M. (1978). Mutagenesis at a specific position in a DNA sequence. Journal of Biological Chemistry, 253(18), 6551–6560. https://doi.org/10.1016/S0021-9258(19)46967-6
Saiki, R. K., Gelfand, D. H., Stoffel, S., et al. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239(4839), 487–491. https://doi.org/10.1126/science.2448875

About the simulations

Each simulator is a simplified version of a published model of cells, signals, instruments or behaviour, and runs in the web browser. The simulations show how the techniques work and how design choices change the results; they do not replace training in a laboratory.

All data are simulated and generated afresh on each run, so results vary between runs, as in real experiments. Nothing is stored or sent: designs and results are lost when the page is closed, unless the protocol is copied or the data file is downloaded.

Last updated 20 October 2026