Microsatellites owe their variability to an increased rate of mutation compared to other neutral regions of DNA. These high rates of mutation can be explained most frequently by slipped strand mispairing (slippage) during DNA replication on a single DNA strand. Mutation may also occur during recombination during meiosis. Some errors in slippage are rectified by proofreading mechanisms within the nucleus, but some mutations can escape repair. The size of the repeat unit, the number of repeats and the presence of variant repeats are all factors, as well as the frequency of transcription in the area of the DNA repeat. Interruption of microsatellites, perhaps due to mutation, can result in reduced polymorphism. However, this same mechanism can occasionally lead to incorrect amplification of microsatellites; if slippage occurs early on during PCR, microsatellites of incorrect lengths can be amplified.
Random microsatellite primers can be developed by cloning random segments of DNA from the focal species. These are inserted into a plasmid or bacteriophage vector, which is in turn implanted into Escherichia coli bacteria. Colonies are then developed, and screened with fluorescently–labelled oligonucleotide sequences that will hybridise to a microsatellite repeat, if present on the DNA segment. If positive clones can be obtained from this procedure, the DNA is sequenced and PCR primers are chosen from sequences flanking such regions to determine a specific locus. This process involves significant trial and error on the part of researchers, as microsatellite repeat sequences must be predicted and primers that are randomly isolated may not display significant polymorphism. Microsatellite loci are widely distributed throughout the genome and can be isolated from semi-degraded DNA of older specimens, as all that is needed is a suitable substrate for amplification through PCR.
Sequences amplified by ISSR-PCR can be used for DNA fingerprinting. Since an ISSR may be a conserved or nonconserved region, this technique is not useful for distinguishing individuals, but rather for phylogeography analyses or maybe delimiting species; sequence diversity is lower than in SSR-PCR, but still higher than in actual gene sequences. In addition, microsatellite sequencing and ISSR sequencing are mutually assisting, as one produces primers for the other.
Microsatellites have proved to be versatile molecular markers, particularly for population analysis, but they are not without limitations. Microsatellites developed for particular species can often be applied to closely related species, but the percentage of loci that successfully amplify may decrease with increasing genetic distance. Point mutation in the primer annealing sites in such species may lead to the occurrence of ‘null alleles’, where microsatellites fail to amplify in PCR assays. Null alleles can be attributed to several phenomena. Sequence divergence in flanking regions can lead to poor primer annealing, especially at the 3’ section, where extension commences; preferential amplification of particular size alleles due to the competitive nature of PCR can lead to heterozygous individuals being scored for homozygosity (partial null). PCR failure may result when particular loci fail to amplify, whereas others amplify more efficiently and may appear homozygous on a gel assay, when they are in reality heterozygous in the genome. Null alleles complicate the interpretation of microsatellite allele frequencies and thus make estimates of relatedness faulty. Furthermore, stochastic effects of sampling that occurs during mating may change allele frequencies in a way that is very similar to the effect of null alleles; an excessive frequency of homozygotes causing deviations from Hardy-Weinberg equilibrium expectations. Since null alleles are a technical problem and sampling effects that occur during mating are a real biological property of a population, it is often very important to distinguish between them if excess homozygotes are observed.
When using microsatellites to compare species,homologous loci may be easily amplified in related species, but the number of loci that amplify successfully during PCR may decrease with increased genetic distance between the species in question. Mutation in microsatellite alleles is biased in the sense that larger alleles contain more bases, and are therefore likely to be mistranslated in DNA replication. Smaller alleles also tend to increase in size, whereas larger alleles tend to decrease in size, as they may be subject to an upper size limit; this constraint has been determined but possible values have not yet been specified. If there is a large size difference between individual alleles, then there may be increased instability during recombination at meiosis. In tumour cells, where controls on replication may be damaged, microsatellites may be gained or lost at an especially high frequency during each round of mitosis. Hence a tumour cell line might show a different genetic fingerprint from that of the host tissue.
Survey of microsatellite clustering in eight fully sequenced species sheds light on the origin of compound microsatellites.(Research article)(Report)
Dec 17, 2008; Authors: Robert Kofler (corresponding author) ; Christian Schlötterer ; Evita Luschützky ; Tamas Lelley  1 Background...