Chemical mutagenesis
Chemical mutagenesis was used to introduce new genetic mutations into
inbred mouse strains. There are several large efforts to dissect complex
traits in the mouse using chemical mutagenesis to produce artificial
mutants, which can be screened for phenotypes of interest (78–80). This
resulted from difficulties encountered by investigators using standard
methods for QTL analysis (reviewed in ref. 50). It was hoped that this
approach would provide a powerful alternative method for complex
trait analysis. This method is attractive because new mutations are
introduced into the genetic background of an inbred mouse strain, and
all genes in the genome are susceptible to mutagenesis. This approach
has been, and will be, useful for identifying highly penetrant genes,
particularly those having a profound effect on embryonic and organ
development (81). A clever strategy has been developed to enable
screening for recessive mutations, but it does require several
generations of intercrossing mutagenized mice (81). However, it is
unlikely that this approach will be generally useful for analysis of
complex genetic traits. Identification of genes with a large phenotypic
effect may not reveal how polygenic traits are regulated. Because most
phenotypic screens performed on mutagenized progeny are not for
quantitative traits, it is unlikely that genes of small effect will be
identified. It is also statistically unlikely that an individual progeny will
have mutations in multiple different loci that will produce quantitative
trait variance. However, mutagenesis programs may produce a very
useful collection of genetic variation on a known background. DNA and
sperm from mutagenized male mice can be archived, and
Computational Biology 23
the archived DNA can be scanned for mutations in selected genes. The
sperm can be recovered from mice with mutations in genes of interest
and used to produce mice for phenotypic studies (82). The application
of an SNP-based genotyping method for analysis of murine intercross
progeny provides another tool that facilitates genetic analysis. An
improved strategy for SNP scoring with allele-specific oligonucleotide
primers and kinetic (real time) monitoring of polymerase chain reaction
(PCR) amplification (83) was utilized for mouse genotyping (65). Allelespecific amplification results from the use of oligonucleotide primers
specific for one or the other SNP variant sequence. This approach
provides an efficient and low-cost method for SNP-allele genotyping.
The genotyping reaction is performed within a single microtiter well
and does not require any post-PCR analytic steps. Most importantly,
this method can determine allele frequencies in pooled DNA samples. A
subset of phenotypically extreme progeny can be selected, and their
DNA can be aggregated into pools. Allele frequency differences between
the pooled samples can be used to identify linkage regions, which
exponentially reduce the amount of genotyping required for analysis of
experimental intercrosses. A web-accessible database, which enables
computational selection of allele-specific primers for genotyping
experimental mouse intercrosses, was established. The oligonucleotide
primer sequences and conditions for performing over 750 allele-specific
kinetic PCR genotyping assays (83) are provided in the mouse SNP
database (see http:\\mouseSNP.roche.com and ref. 65).