5.6.2 Random Mutagenesis
Random mutagenesisis a method of introduction of multiple mutations into a DNA sequence but in arbi-
trary sequence positions. Random mutagenesis was achieved classically by reaction of DNA with chemicals
(Chapter 8) to establish its biological role. More recently, random mutagenesis has been used to evolve
DNA sequences that code for proteins with new or enhanced properties and in the evolution of new DNA
catalysts (Section 5.7.3). The introduction of a number of random mutations into a DNA sequence gener-
ates a library of new DNA entities, which can then be used to identify those sequences with the required
functionality. There are two main methods of random mutagenesis.
In error-pronePCR, the aim is to modify the usual PCR protocol (Section 5.2.2) in order to deliberately
introduce mutations. There are several ways in which this may be done. For example, the use of a poly-
merase which lacks proof-reading ability (such as TaqDNA polymerase) allows errors inherent in DNA
synthesis to go uncorrected. Other changes to help reduce the fidelityof the polymerase include a lower
annealing temperature in the PCR cycle, low or unequal dNTP concentrations and/or use of a large num-
ber of PCR cycles (60–80), which allows amplification of erroneous copies. Another common method of
increasing polymerase infidelity is to increase the Mg^2 concentration (up to 10 mM) in the PCR reaction
or to replace Mg^2 by Mn^2 (typically 0.05–0.5 mM). Mutated products can be amplified by further
rounds of PCR.
A second method for introduction of mutations is the use of nucleotide analoguesinto the nascent DNA
during PCR. When the nucleotide analogue is copied in subsequent rounds of PCR, it is not recognised by
the polymerase as a normal nucleotide and an incorrect deoxyribonucleotide is inserted opposite the analogue.
Examples of such analogues includes 2-deoxyinosine, 5-fluoro-2-deoxyuridine, 8-oxo-2-deoxyguano-
sine and the degenerate pyrimidine analogue dP (Figure 5.19).
Nucleic Acids in Biotechnology 191
Figure 5.18 The creation of DNA deletions by PCR