A porcine minisatellite located on Chromosome 14q29

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Microsatellites or short tandem repeats (STR) have undoubtedly become the most successful class of anonymous genetic markers. They can be typed very efficiently by means of PCR (Weber and May 1989; Litt and Luty 1989) and are almost randomly distributed in mammalian genomes (Stallings et al. 1991; Winterr et al. 1992). In the pig genome, they are underrepresented in the telomeric regions (Winterr et al. 1992). Minisatellites, on the other hand, have become less attractive in linkage studies. This is not only owing to the laborious typing by Southern blot hybridization, but also because there are indications for a nonrandom distribution in the human genome towards proterminal regions (Royle et al. 1988). However, highly polymorphic minisatellites located at the telomeres could complement a microsatellite-based map, as end points for chromosomes. Thus far, only two porcine minisatellites have been described (Coppieters et al. 1990; Davies et al. 1992). Here we report the isolation, partial sequencing, and localization of a new pig minisatellite locus. A size-selected library containing genomic pig DNA fragments ranging from 3 to 4.5 kb was constructed in pUC 18:500 ~tg genomic DNA was digested to completion with MboI and separated on a 0.8% agarose gel. The 2 to 23-kb fragments were electroeluted and subsequently concentrated to 30 ~tl by ethanol precipitation. This fraction was separated a second time on a 1% Seaplaque agarose gel (FMC, Rockland, USA). The agarose was sliced and digested with Gelase (Epicentre Technologies, Madison, Wis., USA). The digested agarose slices that contain the right-sized fragments were used in a ligation reaction with BamHI-digested and dephosphorylated pUC 18 vector. The ligation mix was transformed in DH5c~ high-efficiency competent E. coli cells (BRL, Gaithersburg, Md., USA) and plated at low density on LB agar plates containing Xgal, IPTG, and ampiciline. A total of 2400 white colonies was transferred to microtiterplates and grown overnight in 150 ~tl Terrific Broth.