Efficient high-resolution genetic mapping of mouse interspersed repetitive sequence PCR products, toward integrated genetic and physical mapping of the mouse genome.

Abstract

The ability to carry out high-resolution genetic mapping at high throughput in the mouse is a critical rate-limiting step in the generation of genetically anchored contigs in physical mapping projects and the mapping of genetic loci for complex traits. To address this need, we have developed an efficient, high-resolution, large-scale genome mapping system. This system is based on the identification of polymorphic DNA sites between mouse strains by using interspersed repetitive sequence (IRS) PCR. Individual cloned IRS PCR products are hybridized to a DNA array of IRS PCR products derived from the DNA of individual mice segregating DNA sequences from the two parent strains. Since gel electrophoresis is not required, large numbers of samples can be genotyped in parallel. By using this approach, we have mapped >450 polymorphic probes with filters containing the DNA of up to 517 backcross mice, potentially allowing resolution of 0.14 centimorgan. This approach also carries the potential for a high degree of efficiency in the integration of physical and genetic maps, since pooled DNAs representing libraries of yeast artificial chromosomes or other physical representations of the mouse genome can be addressed by hybridization of filter representations of the IRS PCR products of such libraries.Mouse genetics is one of the most powerful tools for the analysis of almost all aspects of vertebrate biology including development, physiology, and pathobiology. Mouse models have already been developed for many human diseases (1-6).

Divergent genetic and epigenetic post-zygotic isolation mechanisms in Mus and Peromyscus

“…For SSLPs, a published subset of markers was used as described previously (Schalkwyk et al, 1999; Table 1). Genotyping via IRS markers is based on the species-specific amplification of IRS-PCR products, owing to divergence in the sequence or presence or absence of repetitive elements surrounding a single-copy DNA sequence (McCarthy et al, 1995). In our case, genotyping was accomplished by hybridizing genetically mapped MSP strain-specific IRS probe DNAs to filters of gridded IRS products of individual backcross fetuses as described previously (Elango et al, 1996).…”

Interspersed repetitive sequence (IRS)-PCR for typing of whole genome radiation hybrid panels

“…For instance, a single primer that anneals to the B1 repeat sequence is able to amplify several thousand different fragments from a mouse genomic DNA template. IRS-PCR was originally developed for the species-specific amplification of sequences from somatic cell hybrids (18), and subsequently served as the technological platform for highthroughput genetic and physical mapping in the mouse (19)(20)(21)(22)(23)(24).…”

Development of the arbitrarily primed-representational difference analysis method and chromosomal mapping of isolated high throughput rat genetic markers

“…We were able to isolate 48 rat IRS-RDA markers that were polymorphic between two rat laboratory strains, ACI͞N (ACI) and BUF͞ Nac (BUF) (7), and all of these 48 markers shared the useful feature of CAAT-RDA markers. The major limitation of the CAAT-RDA and IRS-RDA method is that the number of markers that can be isolated is not sufficient (3,6,7). Because the genomic regions represented by CAAT-PCR and IRS-PCR are limited by the primer sequence, a limited number of polymorphic fragments exist in the represented genomic regions.…”

“…Because the DNA fragments present in the CAAT-amplicon were highly enriched, we did not need to concentrate the PCR solution before dot-blotting. We also applied interspersed repetitive sequence (IRS)-RDA, which originally was developed in the mouse (6), to the rat. We were able to isolate 48 rat IRS-RDA markers that were polymorphic between two rat laboratory strains, ACI͞N (ACI) and BUF͞ Nac (BUF) (7), and all of these 48 markers shared the useful feature of CAAT-RDA markers.…”