Microsatellite

Prominent early applications include the identifications by microsatellite genotyping of the eight-year-old skeletal remains of a British murder victim (Hagelberg et al. 1991), and of the Auschwitz concentration camp doctor Josef Mengele who escaped to South America following World War II (Jeffreys et al.

Microsatellites in non-coding regions may not have any specific function, and therefore might not be selected against; this allows them to accumulate mutations unhindered over the generations and gives rise to variability that can be used for DNA fingerprinting and identification purposes.

[6] In white blood cells, the gradual shortening of telomeric DNA has been shown to inversely correlate with ageing in several sample types.

[20] Thus, slippage changes in repetitive DNA are three orders of magnitude more common than point mutations in other parts of the genome.

A study comparing human and primate genomes found that most changes in repeat number in short microsatellites appear due to point mutations rather than slippage.

Others are located in regulatory or even coding DNA – microsatellite mutations in such cases can lead to phenotypic changes and diseases.

For example, microsatellite length changes are common within surface membrane proteins in yeast, providing rapid evolution in cell properties.

[39] Length changes in short sequence repeats in a fungus (Neurospora crassa) control the duration of its circadian clock cycles.

The human genome contains many (>16,000) short sequence repeats in regulatory regions, which provide 'tuning knobs' on the expression of many genes.

[30][41] Length changes in bacterial SSRs can affect fimbriae formation in Haemophilus influenzae, by altering promoter spacing.

[41] Microsatellites in control regions of the Vasopressin 1a receptor gene in voles influence their social behavior, and level of monogamy.

[43] In addition, other GGAA microsatellites may influence the expression of genes that contribute to the clinical outcome of Ewing sarcoma patients.

For example, a GAA triplet expansion in the first intron of the X25 gene appears to interfere with transcription, and causes Friedreich's ataxia.

It is theorized that these sequences form highly stable cloverleaf configurations that bring the 3' and 5' intron splice sites into close proximity, effectively replacing the spliceosome.

Microsatellites are widely used for DNA profiling, also known as "genetic fingerprinting", of crime stains (in forensics) and of tissues (in transplant patients).

In tumour cells, whose controls on replication are 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, and, especially in colorectal cancer, might present with loss of heterozygosity.

[54][55][56] Genome Wide Association Studies (GWAS) have been used to identify microsatellite biomarkers as a source of genetic predisposition in a variety of cancers.

[60] It is used for the genetic fingerprinting of individuals where it permits forensic identification (typically matching a crime stain to a victim or perpetrator).

[61] The microsatellites in use today for forensic analysis are all tetra- or penta-nucleotide repeats, as these give a high degree of error-free data while being short enough to survive degradation in non-ideal conditions.

During the 1990s and the first several years of this millennium, microsatellites were the workhorse genetic markers for genome-wide scans to locate any gene responsible for a given phenotype or disease, using segregation observations across generations of a sampled pedigree.

[66] A microsatellite with a neutral evolutionary history makes it applicable for measuring or inferring bottlenecks,[67] local adaptation,[68] the allelic fixation index (FST),[69] population size,[70] and gene flow.

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.

[75][76] Microsatellites can be analysed and verified by established PCR amplification and amplicon size determination, sometimes followed by Sanger DNA sequencing.

Microsatellites can be amplified for identification by the polymerase chain reaction (PCR) process, using the unique sequences of flanking regions as 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.

These random segments are inserted into a plasmid or bacteriophage 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.

[21][83] 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.

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.