Editing Microsatellite (section)

===Design of microsatellite primers===
If searching for microsatellite markers in specific regions of a genome, for example within a particular [[intron]], 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, 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 random segments 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 hybridize 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="Jarne 1996" /><ref name="Queller">{{cite journal | vauthors = Queller DC, Strassmann JE, Hughes CR | title = Microsatellites and kinship | journal = Trends in Ecology & Evolution | volume = 8 | issue = 8 | pages = 285–8 | date = August 1993 | pmid = 21236170 | doi = 10.1016/0169-5347(93)90256-O }}</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.

More recent techniques involve using oligonucleotide sequences consisting of repeats complementary to repeats in the microsatellite to "enrich" the DNA extracted ([[microsatellite enrichment]]). The oligonucleotide probe hybridizes with the repeat in the microsatellite, and the probe/microsatellite complex is then pulled out of solution. The enriched DNA is then cloned as normal, but the proportion of successes will now be much higher, drastically reducing the time required to develop the regions for use. However, which probes to use can be a trial and error process in itself.<ref name="Kaukinen">{{cite journal |vauthors=Kaukinen KH, Supernault KJ, and Miller KM |year=2004 |title=Enrichment of tetranucleotide microsatellite loci from invertebrate species |journal=Journal of Shellfish Research |volume=23 |issue=2 |page=621}}</ref>