Environmental Engineering Reference
In-Depth Information
Gene expression experiments can also be performed by hybridizing a single labeled
mRNA sample to “macroarrays” of DNA elements that are supported on positively charged
filters. Specialty arrays can be made and analyzed by this method relatively cheaply, and
human, mouse, and microbial macroarrays are commercially available (SigmaGenosys, The
Woodlands, TX; Research Genetics, Huntsville, AL; Clontech Laboratories, Palo Alto, CA;
Genome Systems, St. Louis, MO). The major disadvantages of this format are reduced
sensitivity, limited numbers of elements, and the need for higher concentrations of labeled
cDNA [12].
Microarray technology is sufficiently promising for medical and pharmaceutical
applications that it is expected to continue to attract strong commercial interest leading to
increasing array element density, greater detection sensitivity, and more cost-effective
methods. Additional details and technical descriptions are available in recent reviews [14,
15].
2.5. Mutagenesis
The DNA molecule consists of two strands of nucleotide bases held together by hydrogen
bonds between complementary bases: adenosine pairs with thymine, and cytosine pairs with
guanine. Within the coding region of a gene, each triplet of nucleotides specifies an amino
acid, and the strand of amino acids in turn folds, sometimes after processing, into a functional
protein. DNA is replicated during each cell division in a growing organism, and although this
process is highly accurate, errors can nevertheless occur in which an incorrect nucleotide is
inserted into the daughter strand. If the incorrect nucleotide is copied faithfully in subsequent
replication, a mutation is generated that may ultimately affect the function of the resulting
protein and give rise to a variant organism [16].
Mutations arise in nature either randomly or as consequences of DNA damage by
ultraviolet (UV) radiation, chemicals, or other mutagenic agents [16]. Researchers seeking
desirable mutations can accelerate the mutagenic process by four primary methods:
stimulating replication error-based mutagenesis with chemicals or radiation; generating
localized sequence alterations through error-prone PCR; inducing combinatorial mutagenesis
through directed evolution; or using structural information about a protein to change specific
amino acids, termed “rational design.” Each of the latter three is conducted in vitro and must
therefore be followed by reintroduction of the mutated sequence into the host organism. If
desired mutations show phenotypes that are recessive to the wild-type phenotype, then the
wild-type gene must first be disabled or displaced by integration of the new sequence in its
place; often, however, the desired mutations have dominant phenotypes (e.g., enabling
function under harsh conditions) so that such measures are not necessary. Nevertheless,
expression of a mutant sequence can be vulnerable to the transcriptional environment at its
locus of integration. In the case that the mutant sequence must be integrated into host DNA to
be maintained stably, numerous transfections may be required before satisfactory expression
is obtained
2.5.1. Chemical and physical mutagenesis
. Chemical and physical agents that damage
DNA induce mutations during subsequent DNA replication. DNA-damaging chemicals
include alkylating agents such as ethylmethanesulfonate and derivatives of nitrosoguanidine
that attach alkyl groups to DNA bases, which promotes mispairing during subsequent
replication. They also include intercalating agents such as ethidium bromide that insert
themselves between base pairs, which changes the spacing between bases and therefore
