Microsatellite DNA: the half-page intro. 
July, 1999

Dave McDonald
Dept. Zoology & Physiology
University of Wyoming
Laramie WY 82071-3166

Microsatellite DNA is a repeated sequence of two to five nucleotides (the A, G, C or T that make up the DNA). The usable repeat lengths are from 8-40 copies--for example AC22 would be a 'necklace' of 22 repeat units of the dinucleotide repeat AC. Microsatellites occur in many places (loci) throughout the genome, but in almost all cases they are in non-coding regions of the DNA. For example, AC22 might occur in thousands of places in the genome. The trick is to find and sequence the flanking regions at a particular place (locus)-the DNA on each side of the repeat. The flanking region is a presumably random (unpatterned) stretch of nucleotides. It should occur at only one locus, because the probability that a longish unpatterned sequence arises more than once is vanishingly small -- P approximately equal to 4-60, if the total length of the flanking regions is 60 base pairs. Because the 'beads' are all the same, mutation can occur relatively easily, by a process called slippage replication. Even if a few 'beads' in the complementary strands of DNA don't match up perfectly, the rest do, and strand 1 can add some base pairs to repair the DNA match, or strand 2 can cut out a few. Thus, a given population might contain variants of ACn, where n is the number of 'beads', with n ranging from, let's say, 10 to 28. If we obtain the sequence information for the flanking regions, we can then design forward and reverse primers for the polymerase chain reaction (PCR). The PCR greatly amplifies the target microsatellite, yielding billions of times more copies than we started with. We then run the PCR-amplified product on an electrophoretic gel, through which fragments of different size travel at different rates. Amplified fragments of the same size (e.g., AC26, AC27) will form bands in the gel that can be visualized by a variety of techniques: we use a highly efficient system of fluorescent labeling that is analyzed by a laser beam in an automated DNA sequencer. The size variants are alleles-variants of the genetic material. At any particular locus, an individual has one band/allele (if it is a homozygote) or two bands (if it's a heterozygote). In the population as a whole, however, there may be many alleles. Typical vertebrate populations can have as many as 5 to 15 alleles at a given microsatellite locus. By analyzing several or many loci, we can perform powerful genetic analyses on the resulting data sets. Potential analyses range from individual identification, to parentage exclusion, to relatedness calculations, to diagnosis of genetically mediated diseases, and on up to analysis of genetic differentiation between populations or species. The mutation rate is relatively high (10-2 to 10-5); for comparison, allozymes have mutation rates of approximately 10-6, meaning one mutation per one million replication events. Because of the high mutation rate, microsatellites are probably not the best molecular marker for higher-level phylogenetic analyses.


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