Environmental Engineering Reference
In-Depth Information
induces insertion of extra nucleotides during replication. Finally, DNA-damaging chemicals
also include bulky adduct- producing agents such as benzo(a)pyrene that attach themselves to
DNA bases, which also promotes mispairing during subsequent replication. Physical
mutagens include electromagnetic radiation such as gamma rays, x-rays, and UV light, as
well as particle radiation such as fast and thermal neutrons as well as alpha and beta particles
[16, 17].
The gene encoding the protein or RNA molecule of interest need not be cloned or even
identified for chemical or physical mutagenesis to be used effectively; indeed, only an activity
of interest is required. Exposure of the experimental organism to the mutagenic conditions is
followed by screening or, ideally, selection for improvement of the trait of interest, where
selection refers to a process that favors reproduction of organisms showing the improved trait
over those without improvement [18]. Improved mutants are typically mated, or back-crossed
to parental strains, to minimize the accumulation of potentially deleterious mutations in non-
target genes, before successive rounds of mutagenesis are undertaken [19].
Chemical and physical mutagenesis methods are relatively straightforward and
inexpensive; however, additional methods have been sought because these methods typically
affect only a few nucleotides at a time and therefore result in limited improvements [20].
2.5.2. Error-prone PCR. Error-prone PCR is widely used to generate random mutants
. In
this approach, the gene of interest has been identified and cloned, and PCR primers are
designed for it. During amplification of the gene, however, a number of changes from normal
PCR are employed: primer annealing temperatures are lowered to diminish fidelity;
nucleotide ratios may be lowered and/or altered from the normal 1:1:1:1 equivalence; a non-
proofreading DNA polymerase is used; high levels of the magnesium (II) ion (Mg
2+
) and
often the manganese (II) ion (Mn
2+
) are used to further diminish replication fidelity; and up to
80 cycles may be conducted [21-23]. Amplified products must then be re-introduced into the
experimental organism and expressed to allow screening or selection for improved mutants
[21, 22]. While this method is rapid, convenient, and generates large numbers of mutants, it
primarily generates “point” mutations, or mutations in which single, isolated nucleotide
changes predominate. As a result, it explores only a small sequence space, meaning it is
unable, through successive rounds of mutagenesis, to converge upon a globally optimal
sequence [20]. Nevertheless, numerous commercial kits are available to facilitate error-prone
PCR, and it remains a popular method of mutagenesis (www.stratagene.com/products/
display;www.jenabioscience.com/images/0ea5cbe470/PP-102.pdf).
2.5.3. Combinatorial mutagenesis
. Combinatorial mutagenesis, in all of its many forms,
attempts to capture the success of the genetic recombination that occurs in sexual
reproduction: the genes of multiple parents are mixed and matched to yield new combinations
that are not present even in the parents.
A common form of combinatorial mutagenesis, though by no means the only form, is
known as DNA shuffling or assembly PCR. In this method, as few as one or as many as 20 or
more parental homologs of the gene of interest are obtained in isolated form, fragmented
randomly with DNase I, and subjected to replication by PCR in the absence of primers.
Prepared this way, the fragments prime each other at their overlapping regions, and
successive rounds of PCR eventually generate full-length products. Ideally, therefore, the
resulting chimeric progeny incorporate sequence elements that have already been selected for
functionality in the parents, while suffering much fewer nonsense, frameshift, and other
nonproductive mutations that are common with random mutagenesis methods. After
