Figure 1. Schematic diagram showing the stages involved in random mutagenesis of cloned DNA. Various permutations are possible. For example, the starting DNA may be a cloned fragment (as illustrated) or a PCR amplification product that is mutated and then cloned.

Figure 2. Site-directed mutagenesis employing a mutagenic oligonucleotide. The mutagenic oligonucleotide has sufficient sequence complementarity to hybridize to the target template DNA, but contains a predefined sequence change (*). Following chain extension and ligation, a heteroduplex results comprising one mutant strand and one wild-type strand. When transformed into bacteria, replication produces homoduplexes of either mutant or wild-type plasmid and colonies are screened for those in which the mutant plasmid has prevailed. dNTPs: deoxynucleoside triphosphates.

Figure 3. Approaches for site-directed mutagenesis using the PCR. (a) Substrate DNA is amplified using the PCR in which one primer contains a predefined sequence change (*). Both strands of the final PCR product contain the mutation: the mutagenic primer (primer B in this illustration) introduces the mutation into one strand and this directs its replication into the complementary strand. In this approach, the location of the mutation is limited by the position of the mutant primer. (b) One approach by which the PCR can be used to introduce a mutation anywhere within a DNA fragment. Primers A and D define the extent of the final mutated fragment. Primers B and C contain the desired sequence change and are complementary. The substrate DNA is amplified with primers A and B in one reaction and primers C and D in a second reaction. The resulting PCR products each contain the mutation in both of their strands. When used in combination in a further PCR containing only primers A and D, cross-hybridization is possible between A–B and C–D strands and the PCR polymerase fills in to yield a full-length A–D fragment that contains the desired mutation in both strands. The relative positions of primers A, B, C and D permit a mutation to be introduced anywhere within a DNA fragment. Several variations on this theme are possible.

Figure 4. Chimeric RNA/DNA oligonucleotides used for directing predefined point mutations into the genome via DNA mismatch repair. (a) Schematic diagram showing the following features of the chimeric oligonucleotides: regions of RNA (dotted lines), regions of DNA (full lines), hairpin loops and the position of the point mutation (*) specified by the chimera. The oligonucleotides form a double-stranded nucleic acid in which the two strands are either complimentary (vertical bars) at all positions except the mutation site or joined by hairpin loops. At the mutation site, the DNA sequence flanked by stretches of RNA contains mutant sequence and the sequence of the complementary strand is wild type. The RNA is believed to function more efficiently during homologous pairing in the nucleus than would DNA. The ribonucleotide precursors used in the synthesis of the RNA stretches are chemically modified to protect against RNases within the cell. This, together with the double-stranded circular structure (the hairpin loops eliminate DNase-sensitive free ends), increases the intracellular survival of the oligonucleotide. (b) An example of a chimeric oligonucleotide containing the features described in (a). Upper case letters represent DNA, lower case letters represent RNA, the targeted point mutation is underlined (A in the wild-type sequence is to be replaced by T).

Figure 5. Approaches for deletion mutagenesis. (a) An exonuclease is used to partially delete a starting fragment of DNA. When cloned, the resulting bacterial colonies comprise a library of deletion fragments of different lengths. This is due to the nucleolytic activity extending further for some molecules of the starting DNA than others during the exonuclease reaction. Depending on the exonuclease used, deletions may be introduced at the 5¢ or 3¢ end of the starting DNA. (b) The PCR can be used to delete a starting fragment of DNA from either or both ends, or to introduce an internal deletion. For both approaches, the extent of the deletion can be specified with absolute precision, unlike deletions introduced using an exonuclease. PCR products containing deletions can either be used directly or cloned (for example, for expression).