Microsatellites

minisatellites consisted of repeats of between 10 and 70, or more, DNA base pairs per repeat block, microsatellites are tandem repeats which have a much simpler structure, consisting of between two and five base pairs per repeat; they are also relatively stable. In contrast to minisatellites, microsatellites are amenable to PCR analysis because the overall length of the tandem repeat is relatively small, seldom exceeding more than a few hundred base pairs. Microsatellites have been widely used for gene mapping. In general, tri- and tetranucleotide repeats are preferred to dinucleotide repeat markers because they produce less confusing secondary bands (satellite bands) on electrophoresis gels. Microsatellites have been the most widely used marker polymorphisms in the Human Genome Project, where they have been used to build detailed linkage maps of all chromosomes; they also form the basis of modern forensic DNA profiling. Although the microsatellite blocks tend to be less polymorphic than the minisatellites in terms of heterozygosity, they are found scattered more evenly throughout the human genome rather than being concentrated in specific regions. The human genome is estimated to contain over one million such loci.Large-scale gene mapping studies frequently involve the analysis of numerous microsatellite loci by gel or capillary electrophoresis. This type of approach can yield several hundred genotypes from a single experiment. Capillary electrophoresis is amenable to automation such that the products of a PCR reaction can be introduced robotically to the electrophoretic instruments. In this case, the microsatellites are amplified using fluorophore-labelled PCR primers and the reaction products are detected by laser-stimulated fluorescence as they pass by a detector that records the electrophoresed DNA fragments. Automated techniques such as capillary electrophoresis can be carried out with relatively little technical support in comparison with the demands of gel electrophoresis. In practical terms, microsatellite analysis can be conduct edusing instruments such as the Applied Biosystems Genescan 3100. The dye-labelled PCR products are loaded on to the instrument and electrophoresed. Specialised software is then used to process and determine the allele sizes of the products and thus the genotypes. Interestingly, some triplet DNA repeat microsatellite sequences have been found to play a key role in the aetiology of some human genetic disorders, particularly those associated with certain inherited neurological conditions such as Huntington disease (HD, OMIM 143100), myotonic dystrophy (DM, OMIM 160900) and fragile X syndrome (FMR1, OMIM 309550). In these cases, the onset and severity of the disorders can be related to the increase in the number of trinucleotide repeats from generation to generation. For example, in HD, an autosomal dominant disease that gives rise to progressive and neural cell death, the onset of disease is associated with increases in the increasing length of a CAG triplet repeat located within the Huntington gene on human chromosome 4. The CAG triplet encodes the amino acid glutamine, which gives rise to a polyglutamine tract within the Huntington gene. It has been noted that in healthy individuals the number of CAGre peats ranges between 10 and 36, whereas in HD patients the number ranges between 37 and 100. The molecular mechanisms leading to microsatellite variation are not completely understood, although microsatellite loci certainly exhibit higher mutation rates compared with single nucleotide substitutions in genes. It is generally accepted that replication slippage is the most common mutational mechanism leading to the gain or loss of one or more repeat units. Other mutational mechanisms such as those based on unequal crossing over or duplication events have also been considered. The tendency to mutate might also depend on the chromosomal environment of a particular locus and whether or not it is transcribed.