Catherine M. Hearne

Microsatellites for linkage analysis of genetic traits

Microsatellites are tandem repeats of simple sequence that occur abundantly and at random throughout most eukaryotic genomes. Since they are usually less than 100 bp long and are embedded in DNA with unique sequence, they can be amplified in vitro using the polymerase chain reaction. Microsatellites are easy to clone and characterize and display considerable polymorphism due to variation in the number of repeat units. This polymorphism is sufficiently stable to use in genetic analyses. Microsatellites are therefore ideal markers for constructing high-resolution genetic maps in order to identify susceptibility loci involved in common genetic diseases.

Additional microsatellite markers for mouse genome mapping

Mouse sequence information from the EMBL and GenBank databases, published sequences and genomic clones have been analyzed for simple repetitive elements or microsatellites. Each microsatellite has been amplified by the polymerase chain reaction (PCR) as a single locus marker. PCR primers were designed from unique sequence flanking each repeat. Size variation of PCR products less than 750 base pairs (bp) between mouse strains has been determined using ethidium bromide-stained acrylamide or agarose gels. A further 74 newly characterized microsatellites are presented in this paper, bringing to 185 the total we have analyzed. Of these, 157/185 (85%) have more than one allele, 143/178 (80%) vary in length between C57BL/6J and Mus spretus, and 82/168 (49%) vary between DBA/2J and C57BL/6J. Microsatellites provide informative single locus probes for linkage analysis in the construction of a genetic map of the mouse genome.

The generation of a library of PCR-analyzed microsatellite variants for genetic mapping of the mouse genome ☆

Forty-three sequences containing simple sequence repeats or microsatellites were generated from an M13 library of total genomic mouse DNA. These sequences were analyzed for size variation using the polymerase chain reaction and gel electrophoresis without the need for radiolabeling. Seventy-two percent of the sequences showed allelic size variations between different inbred strains of mouse and the wild mouse, Mus spretus; and 53% showed variation between inbred strains. Thirty-seven percent were variant between B6/J and DBA/2J, and 81% of these were resolved using minigel agarose electrophoresis alone. This approach is a useful way of generating the large number of variants that are needed to create high resolution maps of the mouse genome.

Mononucleotide repeats are an abundant source of length variants in mouse genomic DNA

Microsatellite sequences, such as dinucleotide repeats, show a high degree of polymorphism in eukaryotic DNA. These sequences are convenient as genetic markers and can be analyzed by the polymerase chain reaction (PCR). We have assessed the frequency of length variants in 18 mononucleotide repeats in mouse DNA and find that the variability is similar to that reported for dinucleotide repeats. Nine of the 18 repeat sequences (50%) have three or more alleles in the strains tested. Ten of these repeat sequences have been mapped using strain distribution patterns (SDPs) in recombinant inbred (RI) strains.

Linkage analysis of 84 microsatellite markers in intra- and interspecific backcrosses.

Microsatellite sequences are stretches of repetitive DNA distributed throughout the genomes of most eukaryotes (Weber and May 1989; Stallings et al. 1991). They are characterized by a short basic unit that is repeated several times in a tandem array. The core unit can be a dinucleotide, trinucleotide, or tetranucleotide. Such sequences have been shown to be highly polymorphic not only between different species of mice, but also between different inbred laboratory strains (Love et al. 1990; Dietrich et al. 1992). Microsatellite length variants are easily detected with the polymerase chain reaction (PCR) provided unique flanking sequence is available for PCR primer design and the resulting PCR products can be resolved by acrylamide/agarose gel electrophoresis. This ease of analysis, coupled with the high degree of polymorphism exhibited by microsatellites, makes them extremely useful markers in the construction of highresolution, easily accessible linkage maps (Love et al. 1990; Dietrich et al. 1992). Such genetic linkage maps have proved invaluable in the mapping of disease susceptibility genes (Todd et al. 1991). The nonobese diabetic (NOD) mouse spontaneously develops diabetes with many similarities to the human disorder type 1 (insulin-dependent) diabetes mellitus and as such is a useful tool for the mapping and identification of diabetes susceptibility loci (Todd et al. 1991). To this end we undertook the construction of a linkage map of the mouse genome, using a series of variant microsatellite markers amplified by PCR and DNA purified from the progeny of two reciprocal first backcross generations (BC1), (BIO.H-2 g7 • NOD)F 1 • NOD and NOD • (BIO.H-2 g7 • NOD)F 1. Between 37 and 471 animals were typed for 94 marker loci distributed over 16 autosomes. The use of a con-