Microsatellites

Microsatellites, or '''Simple Sequence Repeats''' (SSRs), are Polymorphic loci present in Nuclear DNA that consist of repeating units of 1-6 base pairs in length. They are typically neutral, Co-dominant and are used as Molecular Marker s which have wide-ranging applications in the field of Genetics , including Kinship and Population studies.

INTRODUCTION

One common example of a microsatellite is a (CA)n repeat, where n is variable between Alleles . These markers often present high levels of inter- and intra-specific polymorphism, particularly when tandem repeats number ten or greater ¹. The repeated sequence is often simple, consisting of two, three or four Nucleotides (di-, tri-, and tetranucleotide repeats respectively), and can be repeated 10 to 100 times. CA nucleotide repeats are very frequent in Human and other Genome s, and present every few thousand base pairs. As there are often many alleles present at a microsatellite locus, Genotype s within Pedigree s are often fully informative, in that the Progenitor of a particular allele can often be identified. In this way, microsatellites are ideal for determining paternity, population genetic studies and Recombination Map ping. It is also the only molecular marker to provide clues about which alleles are more closely related ².

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 Double Helix . Mutation may also occur during Recombination during Meiosis ³. 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.

AMPLIFICATION OF MICROSATELLITES

Microsatellites can be amplified for identification using Polymerase Chain Reaction ( PCR ), using templates of flanking regions (primers). DNA is denatured at a high temperature, separating the double strand, allowing annealing of Primers and the extension of nucleotide sequences along opposite strands at lower temperatures. This process results in production of enough DNA to be visible on Agarose or Acrylamide gels; only small amounts of DNA are needed for amplification as thermocycling in this manner creates an exponential increase in the replicated segment ⁴. With the abundance of PCR technology, primers that flank microsatellite loci are simple and quick to use, but the development of such primers is often a tedious and costly process.

Development of Microsatellite Primers

Microsatellite primers are developed by Cloning random segments of DNA from the focal species. These are inserted into a Plasmid or Phage 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 ⁵. 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.

LIMITATIONS OF MICROSATELLITES

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 ⁶. 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 falsely Homozygous . However, Stochastic effects of small populations and the possibility of sex linkage must also be considered in order not to give false evidence of a null allele due to increased homozygosity within population analysis. Allele size differences may not reflect true divergence i.e. mutation may result from addition or deletion of bases and overall microsatellites may be under certain constraints in length. Mutation rates are not standard, and the neutrality of some microsatellite regions are coming under question, perhaps due to Quantitative Trait variation Ellegren, 2001 or occurrence within exon regions of genes under selection . 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, the 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.

SEE ALSO

Minisatellite , Genetic Marker , Mobile Element , Transposon , Short Interspersed Repetitive Element , Long Interspersed Repetitive Element , Junk DNA , Variable Number Tandem Repeat s, Short Tandem Repeat s, Trinucleotide Repeat Disorders

REFERENCES

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Microsatellites