On each side of the repeat unit are flanking regions that consist of "unordered" DNA.
The flanking regions are critical because they allow us to develop locus-specific primers to amplify the microsatellites with PCR (polymerase chain reaction).
PUC19 is a commonly used plasmid for this sort of analysis. Criteria that enter into primer selection include:e) avoidance of repeats near end of sequenced region.
Some of the positive clones we have sequenced may have good repeat units, but be too close to the end of the sequence.
The primers for PCR will be sequences from these unique flanking regions. The reason seems to be that their mutations occur in a fashion very different from that of "classical" point mutations (where a substitution of one nucleotide to another occurs, such as a G substituting for a C).
By having a forward and a reverse primer on each side of the microsatellite, we will be able to amplify a fairly short (100 to 500 bp, where bp means base pairs) locus-specific microsatellite region. The mutation process in microsatellites occurs through what is known as slippage replication.
They can be used to assess demographic history (e.g., to look for evidence of population bottlenecks), to assess effective population size (N) and to assess the magnitude and directionality of gene flow between populations.
Microsatellites provide data suitable for phylogeographic studies that seek to explain the concordant biogeographic and genetic histories of the floras and faunas of large-scale regions. First, the microsatellite primer sites may not be conserved (that is the primers we use for Species A may not even amplify in Species B).
We would send out an order for the primer sequences (in our case we add an additional 19 bp M13 tail, which allows us to attach fluorescent nucleotides/d NTPs to our amplified product in the PCR).
A laser in our sequencer/automated genotyper then detects the fluorescence, which is how we visualize the bands that constitute the allelic data we hope to gather and analyze.
They have also become the primary marker for DNA testing in forensics (court) contexts -- both for human and wildlife cases (e.g., Evett and Weir, 1998).
The reason for this prevalence as a forensic marker is their high specificity.
, where the two nucleotides A and C are repeated in bead-like fashion a variable number of times (n could range from 8 to 50).