Microsatellites

Description

Microsatellites, like minisatellites, represent tandem repeats, but their repeat motifs are shorter (1-6 base pairs). If nucleotide sequences in the flanking regions of the microsatellite are known, specific primers (generally 20-25 bp) can be designed to amplify the microsatellite by PCR. Microsatellites and their flanking sequences can be identified by constructing a small-insert genomic library, screening the library with a synthetically labelled oligonucleotide repeat and sequencing the positive clones. Alternatively, microsatellites may be identified by screening sequence databases for microsatellite sequence motifs from which adjacent primers may then be designed. In addition, primers may be used that have already been designed for closely related species. Polymerase slippage during DNA replication, or slipped strand mispairing, is considered to be the main cause of variation in the number of repeat units of a microsatellite, resulting in length polymorphisms that can be detected by gel electrophoresis. Other causes have also been reported. Synonyms used for microsatellites include Simple Sequence Length Polymorphisms (SSLP), Simple Sequence Repeats (SSR) and Sequence Tagged Microsatellites (STMS).

Strengths

The strengths of microsatellites include the codominance of alleles, their high genomic abundance in eukaryotes and their random distribution throughout the genome. Because the technique is PCR-based, only low quantities of template DNA are required. Due to the use of long PCR primers, the reproducibility of microsatellites is high and analyses do not require high quality DNA. Although microsatellite analysis is, in principle, a single-locus technique, multiple microsatellites may be multiplexed during PCR or gel electrophoresis if the size ranges of the alleles of different loci do not overlap. This decreases significantly the analytical costs. Furthermore, the screening of microsatellite variation can be automated.

Weaknesses

One of the main drawbacks of microsatellites is that high development costs are involved if adequate primer sequences for the species of interest are unavailable, making them difficult to apply to unstudied groups. Although microsatellites are in principle codominant markers, mutations in the primer annealing sites may result in the occurrence of null alleles (no amplification of the intended PCR product), which may lead to errors in genotype scoring. The potential presence of null alleles increases with the use of microsatellite primers generated from germplasm unrelated to the species used to generate the microsatellite primers (poor “cross-species amplification”). Null alleles may result in a biased estimate of the allelic and genotypic frequencies and an underestimation of heterozygosity. Furthermore, the underlying mutation model of microsatellites (infinite allele model or stepwise mutation model) is still under debate. Homoplasy may occur at microsatellite loci due to different forward and backward mutations, which may cause underestimation of genetic divergence. A very common observation in microsatellite analysis is the appearance of stutter bands that are artifacts in the technique that occur by DNA slippage during PCR amplification. These can complicate the interpretation of the band profiles because size determination of the fragments is more difficult and heterozygotes may be confused with homozygotes. However, the interpretation may be clarified by including appropriate reference genotypes of known band sizes in the experiment.

Applications

In general, microsatellites show a high level of polymorphism. As a consequence, they are very informative markers that can be used for many population genetics studies, ranging from the individual level (e.g. clone and strain identification) to that of closely related species. Conversely, their high mutation rate makes them unsuitable for studies involving higher taxonomic levels. Microsatellites are also considered ideal markers in gene mapping studies.

Description

Strengths

Weaknesses

Applications

Suggested reading