Like rRNA genes, most VNTR loci are concentrated in proterminal regions of human chromosomes (but not mouse chromosomes) and thus may not be able to provide the desired density of markers. This problem is largely overcome in mi microsatellite or SSR loci described in the following text.

In 1989, several laboratories also used PCR to demonstrate a high level of polymorphism or allelic variation in the repeat number (10-50) of SSRs (simple sequence repeats, usually 1-6bp long), known as 'microsatellites', Microsatellites occur frequently and randomly everywhere in all eukaryotic DNAs examined except yeast, and are so named by analogy with the larger minisatellite arrays or VNTRs as above.

Genomic DNA is digested with a frequent cutting enzyme (Sau3AI, or a combination of Alul, Rsal and HaeIII) and fragments are cloned in sequencing vectors. Genomic libraries generated thus are screened for the presence of microsatellites by hybridization with simple repetitive oligonucleotide probes, e.g. (AC)n, (AAG)n, etc.

Positive clones are then sequenced to find out the flanking sequences to be used as primers for PCR analysis. For libraries of larger insert size, a sequencing primer may be annealed directly to the repeat, to determine one of the flanking

To be useful for construction of linkage maps, microsatellites should generally have 4-5 alleles and should also have PIC (polymorphic information content) > 0,7.