Figure 1. (a) Unfolded and fully solvated polypeptide chain; (b) folded chain, right. Large green circles represent hydrophobic side-chains; red circles represent oxygen; blue circles represent hydrogen. Arrow and coil represent ordered regions of the folded protein: helix and strand. Note that, in the unfolded state, both the backbone and side-chain atoms of protein interact with water molecules and in the folded state they are secluded from water by forming hydrophobic packing and internal hydrogen bonds.

Figure 2. A wild-type plasmid (upper left) is used as a template for producing a plasmid with a specified mutation at a particular site. After temperature-induced denaturation, each primer strand binds the complementary single strand of the plasmid DNA. In the presence of polymerase, DNA replication proceeds from the 3¢ end of the primer, producing two mutant plasmids, each having an unligated site (|) where replication terminates. That completes the first cycle. Another round of heating produces unmutated single-stranded circular DNA and mutated linear DNA. Reannealing produces primer bound to unmutated DNA, which serves as template for another mutated strand, and inert double-stranded mutant DNA. If there are N plasmids initially, each round produces 2N mutant strands, with 2rN mutant strands after r rounds.

Figure 3. Production of mutant plasmids by cassette synthesis and combinatorial ligation. A gene (blue line) is excised and fragmented, in the example shown into three pieces, and different mutant codons are introduced (coloured circles) into the fragments. If codons are introduced at equal frequency, then the probability is 1/64 that a randomly picked fragment will have a particular codon. Fragments are selected in random triplets for ligation to reform an intact and triply mutated gene. The example shown can produce more than 2.6 × 10⁵ different mutants.

Figure 4. The generation of sequence diversity by shuffling and error-prone PCR. The genes are randomly fragmented into strands of different length. Overlapping fragments will bind one another, and the single-stranded 3¢ end of the duplex will serve as template for continuation of the 3¢ end of the bound partner, adding nucleotides without error correction. The resulting strands now differ from the wild type by a small fraction of point mutations, and by random recombination. Another round of random fragmentation and PCR is followed by selection of desired mutants (for example increased affinity for some substrate) and repetition of the cycle on the selected strands.

Figure 5. Differential free energy changes in double mutant cycles.