Microsatellites

What is Microsatellites ?
Microsatellites, also known as Simple Sequence Repeats (SSRs) or short tandem repeats (STRs). A specific sequence of DNA bases (nucleotides). Locations : nuclear DNA , chloropalst

Type of Microsatellites
1. Dinucleotide
Animals - CA Plants - TA, GA

2. Trinucleotide
GTG, CAG, and AAT Related to disease and cancers

3. Tetranucleotide
GATA/GACA Highly polymorphic.

Microsatellites
At a given microsatellite, different individuals can have different numbers of repeats. Changes in the number of repeats result from mutation. Microsatellites mutate very rapidly (100 10,000 times faster than normal base pair substitutions), so there is lots of variation between individuals.
ATATATATAT ATATATATATATAT = 5 repeats = 7 repeats

Individuals have two copies of every microsatellite (one from Mom and one from Dad). The copies can be the same or different.
CGACGACGACGA CGACGACGACGACGA ATATATATAT ATATATATAT

Heterozygous

Homozygous

Detection of Microsatellites
The most common way to detect microsatellites is to design PCR primers that are unique to one locus in the genome and that base pair on either side of the repeated portion (figure 1). Therefore, a single pair of PCR primers will work for every individual in the species and produce different sized products for each of the different length microsatellites.

 Figure 1. Detecting microsatellites from genomic DNA. Two PCR primers (forward and reverse gray arrows) are designed to flank the microsatellite region. If there were zero repeats, the PCR product would be 100 bp in length. Therefore, by determining the size of each PCR product (in this case 116 bp), you can calculate how many CA repeats are present in each microsatellite (8 CA repeats in this example).

 The number of repeats can be determined by separating microsatellites by size using electrophoresis. Gel Electrophoresis Alleles are named based on their size: at a locus where -AT is repeated, for example, possible alleles include 60, 62, 64.

Capillary Electrophoresis

Microsatellites
Microsatellites are junk DNA. In humans, 90% of microsatellites are found in noncoding regions of the genome. Microsatellites may provide a source of genetic variation. In bacteria, variation in microsatellites alleles in coding regions is thought to be adaptive in different environments. Microsatellites may help regulate gene expression.

Microsatellite Advantages
1. 2. 3. 4. 5. 6. Highly Polymorphic Codominant In every organism examined to date Very abundant Random spacing in the genome Can find same loci in closely related species 7. Easy and reliable scoring 8. Highly sensitive 9. Neutral markers

Microsatellite Disadvantages
1. Expensive 2. Time consuming 3. Several loci are needed to obtain sufficient statistical power 4. Current analyses methods do not distinguish between changes in flanking regions vs. changes within the microsatellite regions 5. Different rates of evolution at different loci

Mutation Mechanisms
1. Slippage in DNA at Replication (Slip-Strand Mispairing, SSM)
increases or decreases the repeat by one unit most supporting evidence

2. Recombination
A. Unequal crossing over (UCO) B. Gene conversion

Microsatellite Mutations

10-3 to 10-6 events per locus per generation (point mutation 10-9 to 10-10) Varies by
repeat type base composition of the repeat taxonomic group

most common - addition or deletion of a single repeat

occasionally 2 to several repeats

strong evidence that the number of repeats is limited

Mutation Models
1. Infinite Allele Model (IAM)
gain or loss of any number of repeats and always results in an allelic state not present in the population

2. Stepwise Mutation Model (SMM)

gain or loss of a single repeat

3. Two-Phase Model (TPM)

gain or loss of X repeats

4. K-allele Model (KAM)

Intermediate step in the IAM (IAM = KAM with infinite K) K possible allelic states

Fluorescent Labeling of Microsatellites

Acrylamide gel with 5 microsatellite loci and internal size standard

Simultaneous analysis of a dozen loci

How do scientists use microsatellites?

Forensics
DNA fingerprinting
Because microsatellites are so variable, by studying several at one time (and getting a DNA fingerprint), individuals can be identified.

Paternity studies
Because individuals receive one allele from their mother and one from their father, paternity (or maternity) can be determined

How do scientists use microsatellites?

Diagnosis and ID of Disease

Microsatellites change in length early in the development of some cancers Useful in linkage studies that try to identify genes associated with certain genetic disorders

Populations Studies
By looking at variation in microsatellites, inferences can be made about population structure.

In this study, we will use microsatellites to:

Determine whether healthy and bleached corals are genetically different If healthy corals have different microsatellite alleles than bleached corals, these microsatellites might be associated with genes that confer resistance to bleaching.

 Microsatellite is distributed along the chromosome arm, in contrast to minisatellites, which are more abundant close to the telomeres in human.

References
Avise, J.C. 1994. Molecular Markers, Natural History and Evolution. Chapman and Hall, New York. 511 pp. Balloux, F. and N. Lugon-Moulin. 2002. The estimate of population differentiation with microsatellite markers. Molecular Ecology. 11: 155-165. Goldstein, D.B. and C. Schloterrer (Editors). 1999. Microsatellites: Evolution and Applications. Oxford University Press, Oxford, 352 pp. Jarne, P and P.J.L. Lagoda. 1996. Microsatellites, from molecules to populations and back. Trends in Ecology and Evolution 11(10): 424-429. Slatkin, M. 1995. A measure of population subdivision based on microsatellite allele frequencies. Genetics 139: 457-462.