Microsatellite

Microsatellites are stretches of DNA that contain series of repeats of a simple sequence motif (typically 1-6 bp), commonly consisting of two or three basepairs. The repetitive nature of these sequences causes the rapid accumulation of mutations, which allows their use as highly variable molecular markers. Such markers find applications in all kinds of fundamental and applied studies within fields such as ecology, forensics and research on genetic diseases. Microsatellites are also known as Simple Sequence Repeats (SSRs) or Short Tandem Repeats (STRs). .

Types and abundance

A number of types of microsatellites can be distinguished varying in complexity and abundance in the genome. A simple example of a microsatellite repeat motif is [CA]₅ which results in the sequence CACACACACA on one strand and of course its complement, [GT]₅, on the other strand. The example is a socalled perfect di-nucleotide repeat. Di-nucleotide repeats are the most common type of microsatellite, while tri- and tetra-nucleotide repeats, such as [CAG]_x and [GATA]_y, also occur frequently. Besides perfect repeats also imperfect repeats exist, which can be seen as derivatives of perfect repeats. An example is [CT]₂[TT][CT]₅, which is a perfect CT repeat with 8 repeats in which the third repeat mutated into TT. Microsatellites have been found in all eukaryote groups and are distributed throughout the nuclear and chloroplast genomes, and are sometimes also found in mitrochondrial DNA. Di-nucleotide repeats are most frequent and on average occur once every 30-50 kbp (kilobasepairs), though sometimes peaking at once every 5 kbp. In animals [CA] repeats are ussually found, while plants are very rich in [TA] or [GA] repeats. Tri-nucleotide repeats are studied mostly in relation to genetic diseases and cancers, because extra repeats do not cause a shift in the reading frame they exist frequently in exons. The diseases are ussually associated with excessively large number of repeats within these loci, sometimes up to a 1000 times the normal length. Examples of diseases in which microsatellites play a role are fragile X syndrome', Friedrich's ataxia and synpolydactyly.

Mutational mechanisms

The use of microsatellites in for instance ecology and forensics depends on their tremendous variability, which allows them to differentiate between very closely related individuals. Microsatellites have been shown to accumulate mutations at rates between 10^-2 (E. coli) and 6 ∙ 10^-6 (Drosophila) events per locus per generation. It has been commonly accepted that the most important mechanism for mutations in microsatellites is what is called strand-slippage mispairing (see ). During DNA replication the template strand and the newly synthesized (nascent) strand can become dissociated as DNA-polymerase enzymes add new bases. Normally this is not a problem, because there is only one way in which the strands can re-anneal. However in stretches of repetitive sequences their are numberous ways in which the strands can re-anneal, which can result in looping out of a number of bases in one strand. These looped out bases cause the two strands to be somewhat mis-aligned, which causes DNA-polymerase to insert or delete a number of repeats in the nascent strand. Usually the difference is only one repeat, but sometimes the change in repeat number is larger.
An other, less frequent, mechanism is uneven crossing-over, whereby parts of alleles cross-over between chromosomes and chromatids. This also occurs because of mis-pairing, but this time the recombination machinary mis-alignes the strands of two chromosomes, which can lead to accumulation of repeats in one strand and deletion in the other, while it can also homogenize their numbers. Unequal crossing-over occurs most easily in very long tandemly repeated sequences.

Functional role

Alltough it is generally assumed that most microsatellite loci occur in non-coding DNA and are selectively neutral, there is evidence that at least some microsatellites have some functional importance. The examples of various associated genetic diseases mentioned above illustrate that there is some restriction on the size that microsatellites can obtain without disrupting the functioning of the organism in which they reside. For instance mutations in microsatellites that do cause shifts in the reading frames of genes may prove to be mal-adaptive and even lethal. Secondly microsatellites are also associated with the regulation of gene-expression, through their protein binding capacity. The deletion, for instance, of microsatellites from certain promotor constructs significantly reduces its transcriptional activity.

Applications

The high mutation rate of microsatellite loci gives them a far greater resolution than more traditional molecular markers such as allozymes. This allows the study of relationships between very closely related populations and individuals. Due to the high number of alleles that can be found in microsatellite loci (different numbers of repeats constitute different alleles) a relatively small number (10-15) of loci is usually required to distinguish between individuals. Microsatellites are analysed through the amplification of loci with locus specific primers in a Polymerase Chain Reaction (PCR). The amplified fragments can be analysed using high resolution (polyacryamide) gel electrophoresis. In the past the locus specific primers were radioactively labelled and visuallized on photo-receptive sheets, while nowadays the primers are usually labelled with a fluorecent dye and analysed in automated DNA-analyzers. These analysers can assess differences in fragment length of a single basepair, allowing very accurate genotyping (in fact these are the machines that are also used for DNA-sequencing). From the fragment lengths the number of repeats at each locus can be determined.

In ecology microsatellites have been applied to estimate various population parameters such as effective population size, inbreeding, gene flow and migration. The ability to differentiate between individuals makes microsatellites excellent markers for parentage analyse and also for suspect identification in forensics. A number of these applications depend on the co-dominant expression of microsatellite alleles. Co-dominant markers, in contast to dominant markers, express all their alleles which allows the identification of homo- and heterozygotes. In microsatellites all alleles can be detected after PCR amplification and fragment sizing. Scoring both homo- and heterozygotes in populations allows the calculation of allele frequencies and thus various analyses that are based on Hardy & Weinberg Equilibrium.

Besides the above advantages the microsatellites´ high mutation rates also cause a few downsides. The most important one being the high probability of homoplasy. In molecular ecological analyses it is assumed that when two alleles are identical that they are identical by decent, which means that the individuals sharing the same alleles are more closely related than individuals with different alleles. However this is not necessarily the case, for example two alleles, one of 15 and one of 17 repeats can both mutate and become 16 repeats each. Now the individuals sharing these 16-repeat alleles do not have alleles that are identical by decent and are thus less closely related than would be inferred when analysing their genotypes using this locus. This can significantly influence the analysis and produce false conclusions. For this reason microsatelites are inappropriate for the comparison of populations that are widely separated in space or time, as the chance of the occurrence of homoplasy increases with time.
A second disadvantage results from the location of microsatellites in rapidly evolving parts of the genome. This means that the locus specific primer sites needed for PCR are rapidly lost and generally a separate set of microsatellite markers must be developed for each species, which can be costly and very time-consuming.