Microsatellite
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{{otheruses4|the DNA sequence|small orbiting spacecraft|Miniaturized satellite}}
'''Microsatellites''', or '''Simple Sequence Repeats''' (SSRs), are [[Polymorphism (biology)|polymorphic]] loci present in [[nuclear DNA|nuclear]] and [[organellar DNA|organellar]] [[DNA]] that consist of repeating units of 1-6 [[base pair]]s in length.<ref name="Turnpenny">{{cite book| author= Turnpenny, P. & Ellard, S.|year=2005|title=Emery's Elements of Medical Genetics, 12th. ed. Elsevier, London}}</ref> 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. Microsatellites can also be used to study gene dosage (looking for [[Gene duplication|duplication]]s or [[genetic deletion|deletion]]s of a particular genetic region).
==Introduction==
One rare 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 one hundred or greater.<ref name="goku">{{cite journal | author=Queller, D.C., Strassman,,J.E. & Hughes, C.R. | year=1993 | title=Microsatellites and Kinship | journal=Trends in Ecology and Evolution|volume=8|pages=285 – 288 | doi=10.1016/0169-5347(93)90256-O}}</ref> 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 are present every few thousand base pairs. As there are often many alleles present at a microsatellite locus, [[genotype]]s within [[Pedigree chart|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.<ref name="Goldstein">{{cite journal | author= D. B. Goldstein, A. R. Linares, L. L. Cavalli-Sforza, and M. W. Feldman | year=1995 | title=An Evaluation of Genetic Distances for Use With Microsatellite Loci | journal=Genetics | volume=139 | pages=463–471}}</ref>
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]].<ref name="Blouin">{{cite journal | author= Blouin, M.S., Parsons, M., Lacaille, V. & Lotz, S. | year=1996 | title=Use of microsatellite loci to classify individuals by relatedness | journal=Molecular Ecology|volume=5|pages=393–401 | doi=10.1111/j.1365-294X.1996.tb00329.x}}</ref> Some errors in slippage are rectified by [[proofreading]] mechanisms within the [[cell nucleus|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 (genetics)|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 by the [[polymerase chain reaction]] (PCR) process, using the unique sequences of flanking regions as [[Primer (molecular biology)|primers]]. DNA is repeatedly denatured at a high temperature to separate the double strand, then cooled to allow annealing of primers and the extension of nucleotide sequences through the microsatellite. This process results in production of enough DNA to be visible on [[Agarose gel electrophoresis|agarose]] or [[acrylamide|polyacrylamide]] gels; only small amounts of DNA are needed for amplification as thermocycling in this manner creates an exponential increase in the replicated segment<ref name="Griffiths">{{cite book| author= Griffiths, A.J.F., Miller, J.F., Suzuki, D.T., Lewontin, R.C. & Gelbart, W.M. | year=1996 | title=Introduction to Genetic Analysis, 5th Edition. W.H. Freeman, New York}}</ref>. With the abundance of PCR technology, primers that flank microsatellite loci are simple and quick to use, but the development of correctly functioning primers is often a tedious and costly process.
===Development of microsatellite primers===
If searching for microsatellite markers in specific regions of a genome; for example within a particular exon of a gene, primers can be designed manually. This involves searching the genomic DNA sequence for microsatellite repeats, which can be done by eye or by using automated tools such as [http://www.repeatmasker.org repeat masker]. Once the potentially useful microsatellites are determined (removing non-useful ones such as those with random inserts within the repeat region), the flanking sequences can be used to design [[oligonucleotide]] primers which will amplify the specific microsatellite repeat in a PCR reaction.
Random microsatellite primers can be developed by [[cloning]] random segments of DNA from the focal species. These are inserted into a [[plasmid]] or [[bacteriophage]] [[Cloning vector|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 (genetics)|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.<ref name="Queller">{{cite journal | author=Queller, D.C., Strassman,,J.E. & Hughes, C.R. | year=1993 | title=Microsatellites and Kinship | journal=Trends in Ecology and Evolution|volume=8|pages=285 – 288 | doi=10.1016/0169-5347(93)90256-O}}</ref><ref name="Jarne">{{cite journal | author= Jarne, P. & Lagoda, P.J.L. | year=1996 | title=Microsatellites, from molecules to populations and back | journal=Trends in Ecology and Evolution | volume=11 | pages=424 – 429 | doi=10.1016/0169-5347(96)10049-5}}</ref> 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.
===ISSR-PCR===
'''ISSR''' (for '''inter-simple sequence repeat''') is a general term for a genome region between microsatellite loci. The complementary sequences to two neighboring microsatelites are used as PCR primers<!-- Theor. Appl. Genet. 89, 998–1006. -->; the variable region between them gets amplified. The limited length of amplification cycles during PCR prevents excessive replication of overly long contiguous DNA sequences, so the result will be a mix of a variety of amplified DNA strands which are generally short but vary much in length.
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<!-- Biota 3, 109–118. --> or maybe delimiting [[species]]<!-- Molecular Phylogenetics and Evolution 37 (2005) 389–401 -->; 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.
==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]].<ref name="Jarne"/> Point mutation in the primer annealing sites in such species may lead to the occurrence of ‘[[null allele]]s’, where microsatellites fail to amplify in PCR assays.<ref name="Jarne"/><ref name="Dakin">{{cite journal | author= Dakin, E.E. & Avise, J.C. | year=2004 | title=Microsatellite null alleles in parentage analysis | journal=Heredity |volume=93|pages=504 – 509 | doi=10.1038/sj.hdy.6800545}}</ref> 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.<ref name="Jarne"/> 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]]
*[[Retrotransposon#SINEs | short interspersed repetitive elements]]
*[[Retrotransposon#LINEs | long interspersed repetitive element]]
*[[junk DNA]]
*[[variable number tandem repeat]]s
*[[short tandem repeat]]s
*[[Trinucleotide repeat disorders]]
*[[microsatellite instability]]
==External links==
*About microsatellites:
** [http://www.bio.davidson.edu/COURSES/genomics/method/microsatellite.html Microsatellite DNA Methodology]
*Search tools :
** [http://tandem.bu.edu/trf/trf.html Tandem Repeats Finder]
** [http://bioinfo.lifl.fr/mreps Mreps]
** [http://atgc.lirmm.fr/star STAR]
** [http://www.sci.brooklyn.cuny.edu/~sokol/tandem TRED]
** [http://strand.imb.ac.ru/swan/index.html TandemSWAN]
** [http://www.biophp.org/minitools/microsatellite_repeats_finder/demo.php Microsatellite repeats finder]
** [http://bioinf.dms.med.uniroma1.it/JSTRING/ JSTRING - Java Search for Tandem Repeats in genomes]
** [http://www.rub.de/spezzoo/cm/cm_phobos.htm Phobos - a tandem repeat search tool for perfect and imperfect repeats - the maximum pattern size depends only on computational power]
**[http://espressosoftware.com/pages/sputnik.jsp sputnik - a simple microsatellite search program]
**[http://pgrc.ipk-gatersleben.de/misa/misa.html MISA - MIcroSAtellite identification tool]
[[Category:Genetics]]
[[Category:Repetitive DNA sequences]]
==References==
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