Demystified ... microsatellites

Abstract

Microsatellite DNA sequences consist of relatively short repeats of one to five base pair units; together with satellites and minisatellites they comprise a larger family known as tandemly repetitive sequences. Microsatellites are found both in prokaryotes and eukaryotes, including humans, wherein they appear scattered almost at random throughout the genome. Although in prokaryotes distinct biological functions have been demonstrated, the role of microsatellites in eukaryotes is less clear. Nevertheless, several interesting hypotheses exist suggesting that certain microsatellites may exert subtle influences on the regulation of gene expression. Although the presence of these subtle mechanisms may be beneficial to a whole population, when they go wrong, as is thought to happen in the case of human trinucleotide repeat associated diseases, such as Huntington's disease, the consequences for the individual can be fatal. Most human microsatellites probably have no biological use at all; however, they are extremely useful in such fields as forensic DNA profiling and genetic linkage analysis, which can be used to search for genes involved in a wide range of disorders. With a primary focus on humans, it is the aim of this review to present an up to date discussion, both of the biological aspects and scientific uses of microsatellite sequences. In the latter case, basic theoretical and technical points will be considered, and as such it may be of use both to laboratory and non-laboratory based readers.

Figure 1

Sequential breakdown of the human genome into component DNA types.

Figure 2

Diagram illustrating the mechanisms of unequal crossover and unequal sister chromatid exchange, which are thought to be responsible for the extreme level of polymorphism seen in many satellite and minisatellite sequences.

Figure 3

Strand slippage during DNA replication. (A) The extending strand slips backwards, resulting in the insertion of an extra repeat unit in the newly synthesised strand. (B) The extending strand slips forwards, resulting in the deletion of a repeat unit in the newly synthesised strand.

Figure 4

Examples of the raw output generated using semi-automated fluorescent technology to genotype microsatellite markers. Here, alleles at two different microsatellite loci that overlap in size range are resolved by labelling with different fluorophores (visualised in blue and green). Amplicons are labelled with their estimated size in base pairs, calculated automatically by comparison with the reference size standards shown in red. These size standards are premixed with the test sample and electrophoresed simultaneously in the same gel lane, automatically controlling for lane to lane variation.

Figure 5

Schematic illustrating a hypothetical microsatellite sequence after PCR amplification. After automated amplicon sizing, the process of allele binning is shown to illustrate the way in which the Mendelian inheritance of microsatellite marker alleles can easily be followed. For simplicity, the microsatellite containing polymerase chain reaction amplicon shown is very small, typically amplicons would be at least twice this size.