Editing Microsatellite (section)

==Analysis==
[[File:Short Tandem Repeat (STR) analysis.png|thumb|300px|Short Tandem Repeat (STR) analysis on a simplified model using [[polymerase chain reaction]] (PCR): First, a DNA sample undergoes PCR with [[Primer (molecular biology)|primers]] targeting certain STRs (which vary in lengths between individuals and their [[allele]]s). The resultant fragments are separated by size (such as [[electrophoresis]]).<ref>Image by Mikael Häggström, MD, using following source image: [https://www.researchgate.net/figure/Principles-of-STR-analysis-STRs-loci-comprise-repetitive-sequences-of-2-7-bp-which-are_fig1_26513043 Figure 1 - available via license: Creative Commons Attribution 4.0 International"], from the following article:<br />{{cite journal|title=Using PCR for molecular monitoring of post-transplantation chimerism|url=https://www.researchgate.net/publication/26513043 |vauthors=Sitnik R, Torres MA, Bacal NS, Rebello Pinho JR|journal=Einstein |location=Sao Paulo |year=2006|volume=4|issue=2 |via=ResearchGate}}</ref>]]
Repetitive DNA is not easily analysed by [[DNA sequencing|next generation DNA sequencing]] methods, for some technologies struggle with [[Polymer#Structure|homopolymeric]] tracts. A variety of software approaches have been created for the analysis or raw nextgen DNA sequencing reads to determine the genotype and variants at repetitive loci.<ref name="Analysis">{{cite journal | author1 = Halman A| author2 = Oshlack A| author-link2 = Alicia Oshlack | title = Accuracy of short tandem repeats genotyping tools in whole exome sequencing data | journal = F1000Research | volume = 9 | pages = 200 | date = 2020 | pmid = 32665844 | doi = 10.12688/f1000research.22639.1 | pmc = 7327730 | s2cid = 213733005 | doi-access = free }}</ref><ref name="Rajan-Babu">{{cite journal | vauthors = Rajan-Babu IS, Peng JJ, Chiu R, Li C, Mohajeri A, Dolzhenko E, Eberle MA, Birol I, Friedman JM | display-authors = 6 | title = Correction to: Genome-wide sequencing as a first-tier screening test for short tandem repeat expansions | journal = Genome Medicine | volume = 13 | issue = 1 | pages = 151 | date = September 2021 | pmid = 34517885 | doi = 10.1186/s13073-021-00961-4 | pmc = 8439056 | s2cid = 256019433 | doi-access = free }}</ref> Microsatellites can be analysed and verified by established PCR amplification and amplicon size determination, sometimes followed by [[DNA sequencing|Sanger DNA sequencing]].

In forensics, the analysis is performed by extracting [[nuclear DNA]] from the cells of a sample of interest, then amplifying specific [[polymorphism (biology)|polymorphic]] regions of the extracted DNA by means of the [[polymerase chain reaction]]. Once these sequences have been amplified, they are resolved either through [[gel electrophoresis]] or [[capillary electrophoresis]], which will allow the analyst to determine how many repeats of the microsatellites sequence in question there are.  If the DNA was resolved by gel electrophoresis, the DNA can be visualized either by [[silver stain]]ing (low sensitivity, safe, inexpensive), or an [[DNA intercalation|intercalating dye]] such as [[ethidium bromide]] (fairly sensitive, moderate health risks, inexpensive), or as most modern forensics labs use, [[Fluorescence|fluorescent dyes]] (highly sensitive, safe, expensive).<ref name="nist">{{cite web |title=Technology for Resolving STR Alleles |url=http://www.cstl.nist.gov/strbase/tech.htm |access-date=2010-09-20}}</ref> Instruments built to resolve microsatellite fragments by capillary electrophoresis also use fluorescent dyes.<ref name="nist"/> Forensic profiles are stored in major databanks. The [[United Kingdom|British]] data base for microsatellite loci identification was originally based on the British [[SGM+]] system<ref>{{cite web |title=The National DNA Database |url=http://www.parliament.uk/documents/post/postpn258.pdf |archive-url=https://web.archive.org/web/20101013140110/http://www.parliament.uk/documents/post/postpn258.pdf |archive-date=2010-10-13 |url-status=live |access-date=2010-09-20}}</ref><ref>{{cite web |title=House of Lords Select Committee on Science and Technology Written Evidence  |url=https://publications.parliament.uk/pa/ld199900/ldselect/ldsctech/115/115we20.htm |access-date=2010-09-20}}</ref> using 10 loci and a [[Amelogenin|sex marker]]. The Americans<ref>{{cite web |title=FBI CODIS Core STR Loci |url=http://www.cstl.nist.gov/strbase/fbicore.htm |access-date=2010-09-20}}</ref> increased this number to 13 loci.<ref name="Butler 2005">{{cite book | vauthors = Butler JM |date=2005|title=Forensic DNA Typing: Biology, Technology, and Genetics of STR Markers, Second Edition |location=New York |publisher=Elsevier Academic Press}}</ref> The Australian database is called the NCIDD, and since 2013 it has been using 18 core markers for DNA profiling.<ref name=":0">{{Cite news |url= https://theconversation.com/from-the-crime-scene-to-the-courtroom-the-journey-of-a-dna-sample-82250 |title=From the crime scene to the courtroom: the journey of a DNA sample | vauthors = Curtis C, Hereward J |date=August 29, 2017|work=The Conversation }}</ref>

===Amplification===
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 (biology)|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 because in this way thermocycling creates an exponential increase in the replicated segment.<ref name="Griffiths">{{cite book |vauthors=Griffiths AJ, Miller JF, Suzuki DT, Lewontin RC, Gelbart WM |year=1996 |title=Introduction to Genetic Analysis |edition=5th |publisher=W.H. Freeman |location=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.[[File:PAGE AgStain Microsat.jpg|thumb|right|A number of DNA samples from specimens of ''[[Littorina plena]]'' amplified using polymerase chain reaction with primers targeting a variable simple sequence repeat (SSR, a.k.a. microsatellite) locus. Samples were run on a 5% polyacrylamide gel and visualized using silver staining.]]

===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>

===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 microsatellites 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===
Repetitive DNA is not easily analysed by [[DNA sequencing|next generation DNA sequencing]] methods, which struggle with homopolymeric tracts.<ref>{{cite journal | vauthors = Tytgat O, Gansemans Y, Weymaere J, Rubben K, Deforce D, Van Nieuwerburgh F | title = Nanopore Sequencing of a Forensic STR Multiplex Reveals Loci Suitable for Single-Contributor STR Profiling | journal = Genes | volume = 11 | issue = 4 | pages = 381 | date = April 2020 | pmid = 32244632 | pmc = 7230633 | doi = 10.3390/genes11040381 | s2cid = 214786277 | doi-access = free }}</ref> Therefore, microsatellites are normally analysed by conventional PCR amplification and amplicon size determination. The use of PCR means that microsatellite length analysis is prone to PCR limitations like any other PCR-amplified DNA locus. A particular concern is the occurrence of '[[null allele]]s':
* Occasionally, within a sample of individuals such as in paternity testing casework, a mutation in the DNA flanking the microsatellite can prevent the PCR primer from binding and producing an amplicon (creating a "null allele" in a gel assay), thus only one allele is amplified (from the non-mutated sister chromosome), and the individual may then falsely appear to be homozygous. This can cause confusion in paternity casework. It may then be necessary to amplify the microsatellite using a different set of primers.<ref name="Jarne 1996"/><ref name="Dakin">{{cite journal | vauthors = Dakin EE, Avise JC | title = Microsatellite null alleles in parentage analysis | journal = Heredity | volume = 93 | issue = 5 | pages = 504–9 | date = November 2004 | pmid = 15292911 | doi = 10.1038/sj.hdy.6800545 | doi-access = free }}</ref> Null alleles are caused especially by mutations at the 3' section, where extension commences.
* In species or population analysis, for example in conservation work, PCR primers which amplify microsatellites in one individual or species can work in other species. However, the risk of applying PCR primers across different species is that null alleles become likely, whenever sequence divergence is too great for the primers to bind. The species may then artificially appear to have a reduced diversity. Null alleles in this case can sometimes be indicated by an excessive frequency of homozygotes causing deviations from Hardy-Weinberg equilibrium expectations.