MS-IRS PCR: a simple method for the isolation of microsatellites.

Microsatellites (MS) are excellent genetic markers because of their prevalence in eukaryotic genomes, ease of typing by PCR and high degree of polymorphism. MS-based genetic maps of the human and mouse genomes have been constructed with average intermarker interval sizes of 1.6 cM and 0.42 cM, respectively (1,6). While these maps are essential resources for gene mapping, their resolution may not be sufficient for positional cloning of disease genes or other genes of interest. For these applications, the generation of chromosomal region-specific novel MS markers is important. The first approach used for the isolation of MS repeats was based on the screening of cloned DNA fragments with radioactively labeled repeat-specific oligonucleotides, followed by sequencing of hybridizing clones. This approach is time consuming and labor intensive, especially when large numbers of clones have to be analyzed. A PCR-based technique, vectorette PCR, has also been adapted for the isolation of MS (4). This method involves the generation of a vectorette-ligated pool of genomic DNA inserts, followed by PCR amplification using MS and vectorette-specific primers to amplify the unique sequences flanking the repeat. While considerably more efficient than hybridization-based screening, vectorette PCR still relies on the construction of several representative vectorette libraries using different restriction enzymes. In addition, because yeast genomic DNA also contains MS repeats, the analysis of yeast artificial chromosomes (YACs) requires pulsed-field gel purification, which can be a major rate limiting step of the procedure. Here, we describe a novel PCRbased method for the isolation of MS from genomic DNA clones. Our approach makes use of the fact that, in addition to MS, mammalian genomes contain various species-specific repeats known as interspersed repetitive sequences (IRS), including Alu in humans and B1 and B2 in the mouse. Using a combination of primers corresponding to MS and IRS sequences, it is possible to amplify genomic regions between the two types of elements (Figure 1). The resulting MS-IRS products are sequenced and, based on the unique sequence obtained, primers are generated to allow the extension of the sequence to the other side of the MS by inverse PCR (see Reference 7 for a schematic diagram of inverse PCR). A major advantage of this technique is the species specificity provided by the IRS primers used in the initial amplification. This specificity permits the analysis of unpurified YAC clones in the presence of yeast genomic DNA. We have applied this technique to isolate novel, polymorphic MS from a mouse YAC clone from the critical chromosomal region of the fatty liver dystrophy (fld) mutation. MS-IRS PCR was performed using Benchmarks