The following protocol uses the microsatellite hybrid capture technique for isolating microsatellite loci [Prochazka 1996, Brown et al., 1995]. It has also benefitted from the protocol developed by the Whitney lab at University of Florida. This technique is a variation of the methods of Kandpal et al. [1994] which uses microsatellite probes attached to magnetic beads to isolates pieces of DNA containing the microsatellites. Briefly, the DNA is cut into small pieces (400-900 bp) using a 4 or 6 base restriction enzyme. Short (20bp) linkers are attached to the DNA, which can later be used to amplify selected fragments in PCR reactions. The DNA is purified and size selected in an agarose gel. The DNA is then denatured to produce single stranded fragments, which can hybridize to microsatellite probe molecules. These probe molecules are biotinylated and the biotin moiety binds irreversibly to streptavidin coated magnetic beads. Application of a small magnet pulls these beads out of solution. Since the microsatellite probes are attached to the beads, and any DNA fragments containing microsatellites are hybridized to these probes, the microsatellite DNA is also pulled out of solution. This 5 minute procedure separates the DNA of interest from the rest of the DNA which does not contain the selected microsatellites. The selected DNA is then amplified using primers designed to the linkers, cloned into a vector, and sequenced. Sequencing of these clones enables primers to be designed for each microsatellite locus.

The microsatellite probes selected for this work was a dinucleotide repeat (CA)16 though tetramer repeats can also be used (such as (GAAA)7 and (GATA)7). These probes have been selected because of their prevalence in other genomes (human [Jurka and Pethiyagoda 1995], fish [Lee and Kocher 1995] and [Waldbieser, 1995]). Tetramers have lower mutation rates and so may also be stable over longer periods of breeding. However, tetramers are less prevalent than dinucleotides.

Carleton KL, Streelman JT, Lee BY, Garnhart N, Kidd M, and Kocher TD. Rapid isolation of CA microsatellites from the tilapia genome. Animal Genetics 33(2): 140-4. (Abstract) (PDF)

References

Brown, J. L.J. Hardwick and A.F. Wright. 1995. A simple method for rapid isolation of microsatellites from yeast artificial chromosomes. Molecular and Cellular Probes 9: 53-58.

Jurk, J. and C. Pethiyagod. 1995. Simple repetitive DNA sequences from primates: compilations and analysis. J. Mol. Evol. 40: 120-126.

Kandpal, R.P., G. Kandpal, S.M. Weissman. 1994. Construction of libraries enriched for sequence repeats and jumping clones, and hybridization selection for region specific markers. Proc. Natl. Acad. Sci. USA 91: 88-92.

Lee, W.-J. and T.D. Kocher. 1996. Microsatellite DNA markers for genetic mapping in Oreochromis niloticus. J. Fish Biology 49: 169-171.

Prochazka, M. 1996. Microsatellite hybrid capture technique for simultaneous isolation of various STR markers. Genome Res. 6: 646-649.

Waldbieser, G.C. 1995. PCR-based identification of AT-rich tri- and tetranucleotide repeat loci in an enriched plasmid library. BioTechniques 19: 742-743.